Ing. Alessia Mazzarotta - unina.itAlessia Mazzarotta Supervisors: Advisor: Prof. Dr. Paolo Antonio...

UNIVERSITA’ DEGLI STUDI DI NAPOLI “FEDERICO II”

PhD thesis in Industrial Products and Process Engineering (XXX cycle)

“ENGINEERED HYDROGEL-BASED MATERIALS FOR

OLIGONUCLEOTIDE DETECTION”

Ing. Alessia Mazzarotta

Supervisors: Advisor:

Prof. Dr. Paolo Antonio Netti Dr. Edmondo Battista Prof. Dr. Filippo Causa

Coordinator:

Prof. Dr. Giuseppe Mensitieri

2014 – 2017

ENGINEERED HYDROGEL-BASED MATERIALS FOR

OLIGONUCLEOTIDE DETECTION

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN

INDUSTRIAL PRODUCTS AND PROCESS ENGINEERING

AUTHOR Ing. Alessia Mazzarotta SUPERVISORS Prof. Dr. P.A. Netti Prof. Dr. F. Causa ADVISOR Dr. Edmondo Battista COORDINATOR Prof. Dr. Giuseppe Mensitieri

I

Table of contents

List of Figures ................................................................................................................. IV

List of Tables ................................................................................................................. VIII

List of Abbreviations ..................................................................................................... IX

Abstract ............................................................................................................................ XI

Chapter 1:

Introduction .......................................................................................... 1

1.1 Hydrogels: principal concepts ....................................................................... 2

1.2 Hydrogels for biosensing ................................................................................ 3

1.3 Biomarkers detection ....................................................................................... 5

1.4 Classifications of hydrogels ............................................................................ 9

1.5 Hydrogels: synthesis procedure .................................................................. 11

1.6 Chemical an physical hydrogel characterization .................................... 15

1.6.1 Swelling behavior.................................................................................. 15

1.6.2 Equilibrium swelling theory ................................................................ 17

1.6.3 Solute diffusion within hydrogels ....................................................... 22

1.7 Aim and outline of dissertation ................................................................... 23

1.8 References ........................................................................................................ 25

Chapter 2:

Core-shell microgels: synthesis and swelling characterization ...... 33

2.1 Introduction ..................................................................................................... 34

2.2 Experimental section ..................................................................................... 36

2.2.1 Materials ................................................................................................. 36

II

2.2.2 Synthesis of core-shell microgels ....................................................... 36

2.2.3 Microgels characterization ................................................................... 37

2.3 Results and discussion ................................................................................... 38

2.3.1 Microgels characterization ................................................................... 38

2.3.2 AFM characterization ........................................................................... 42

2.3.3 Swelling characterization ..................................................................... 45

2.4 Core-shell applications as biosensor .......................................................... 48

2.5 Conclusions ...................................................................................................... 49

2.6 References ........................................................................................................ 51

Chapter 3:

PEGDA hydrogels as potential engineered-based assay ................ 53

3.1 Introduction ..................................................................................................... 54


3.2.1 Materials ................................................................................................. 57

3.2.2 Synthesis of bulk-hydrogel .................................................................. 57

3.2.3 Bulk-hydrogel characterizations ......................................................... 60


3.3.1 Swelling characterization ..................................................................... 63

3.3.2 NMR: diffusion studies ........................................................................ 64

3.3.3 Preliminary study for the assay ........................................................... 73

3.4 Conclusions ...................................................................................................... 80

3.5 Appendix A ...................................................................................................... 81

3.6 Appendix B ...................................................................................................... 82

3.7 References ........................................................................................................ 84

III

Chapter 4:

Engineered PEGDA-microparticles by microfluidics:

oligonucleotide functionalization for selective

miRNA 143-3p detection in serum .................................................... 87

4.1 Introduction ..................................................................................................... 88

4.1.1 Microfluidic technique ......................................................................... 88

4.1.2 Biomarkers detection ............................................................................ 91


4.2.1 Materials ................................................................................................. 93

4.2.2 Synthesis of functionalized microparticles ........................................ 94

4.2.3 Microparticles characterization ........................................................... 96


4.3.1 Probe design ........................................................................................... 98

4.3.2 Microparticles: optimization of microfluidic parameters .............. 100

4.3.3 Microparticles: morphological characterization ............................. 102

4.3.4 Microparticles: swelling characterization ........................................ 103

4.3.5 Diffusion studies: CLSM analysis .................................................... 106

4.3.6 miRNA 143-3p detection ................................................................... 111

4.4 Conclusions .................................................................................................... 116

4.5 References ...................................................................................................... 117

Chapter 5:

Conclusions and future perspectives .............................................. 121

IV

List of Figures

Figure 1.1: Hydrogels with tunable control of mesh size ...................................... 1

Figure 1.2: Histogram showing the increase in publications related to the keyword “hydrogel” during the past 50 years.. .................................................. 2

Figure 1.3: Schematic illustration of biosensor. .................................................... 3

Figure 1.4: Chemical structures of PEG and its di(meth)acrylate. ........................ 4

Figure 1.5: Most common biomarkers. .................................................................. 5

Figure 1.6: Compartmentalization of circulating miRNAs. ................................. 7

Figure 1.7: Conventional techniques for miRNAs detection. ................................ 8

Figure 1.8: Principal enviromental factors affecting hydrogels size. .................. 10

Figure 1.9: Different microfluidic techniques ..................................................... 13

Figure 1.10: A crosslinked hydrogel structure with the main swelling parameters ........................................................................................ 16

Figure 1.11: Parameters which influence diffusion of probe particles in polymeric media. ............................................................................... 23

Figure 2.1: Schematic representation of core-shell microgel synthesis obtained combining precipitation and seeded polymerizations. ...................... 39

Figure 2.2: DLS analysis: particle size distribution of R1-core........................... 40

Figure 2.3: DLS analysis: particle size distribution of 1st shell R1PEG. ............. 40

Figure 2.4: DLS analysis: particle size distribution of 2nd shell R1F1AA100. ....... 41

Figure 2.5: DLS analysis: Zeta potential distribution of 2nd shell R1F1AA100. .... 41

Figure 2.6: (a) AFM images of second shell microgels in dry, relaxed and swollen state. (b) microgels profile taken in three different direction along one microgel in dry, relaxed and swollen state. ....................................... 43

V

Figure 2.7: Swelling ratio calculated in both swollen and relaxed state for microgels (Qs, Qr) and their crown (Qs*, Qr*) .................................. 45

Figure 2.8: Focus on the overlap layer between shells. ....................................... 46

Figure 2.9: Graphical representation of core double shell with mechanism of miRNA detection. .............................................................................. 49

Figure 3.1: Schematic representation of engineered bulk hydrogels. .................. 55

Figure 3.2: UV free radical photopolymerization ................................................ 58

Figure 3.3: UV free radical photopolymerization between PEGDA and methacrylate oligonucleotide. ........................................................... 59

Figure 3.5: DOSY sequence ................................................................................ 62

Figure 3.6: Mesh size values for different bulk-PEGDA concentrations. ........... 64

Figure 3.7: 1H-NMR spectrum of PEGDA/darocur solution before polymerization with signal attribution and related peak integration. ......................... 65

Figure 3.8: 1H-NMR spectrum of PEGDA/darocur solution after polymerization. ........................................................................................................... 66

Figure 3.9: 2D DOSY of water in hydrogels bulk with different PEGDA concentrations. ................................................................................... 67

Figure 3.10: Diffusion coefficient versus water content for hydrogels sample. .... 68

Figure 3.11: Probe diffusion coefficient as function of its molecular weight. ...... 69

Figure 3.12: Diffusion coefficient of several probes in both water and hydrogels with different PEGDA concentrations. ............................................. 70

Figure 3.13: Effect of the polymer concentration on the reduction of diffusion coefficients (D/D0). ........................................................................... 71

Figure 3.14: Diffusion coefficients of dextrans probes as function of their hydrodynamic radius obtained in water and PEGDA bulk ............... 72

Figure 3.15: Scheme of oligonucleotide detection. ............................................... 73

Figure 3.16: Quenching percentage in solution for different oligonucleotide concentrations. ................................................................................... 74

Figure 3.17: ATTO 647 response to UV treatment, with and without darocur in solution. ............................................................................................. 74

VI

Figure 3.18: Fluorescence intensity of ATTO-BHQ in PEGDA 20% and 10% . . 75

Figure 3.19: Quenching percentage with both complementary (C-BHQ) and several not complementary concentrations (N-BHQ). .................................. 76

Figure 3.20: Quenching kinetic in PEGDA 10% with several oligonucleotides concentrations. ................................................................................... 77



Figure 4.1: Droplet generation in three microfluidic devices: T-junction, flow focusing and co-flowing geometry.. .................................................. 89

Figure 4.2: Schematic of flow regimes in T-junction microfluidic devices in both dripping (a) and jetting (b) regime. ................................................... 90

Figure 4.3: Engineered microparticles for specific target detection. ................... 91

Figure 4.4: Histogram showing the increase in publications related to the keyword “microRNA” during the past 20 years. ............................................. 91

Figure 4.5: The Droplet Junction Chip and its Chip Interface H for fluidic connection. ........................................................................................ 94

Figure 4.6: Dolomite chip: geometry and size. .................................................... 95

Figure 4.7: Set-up for functionalized microparticles generation. ........................ 96

Figure 4.8: New scheme showing the mechanism of miRNA detection based on ds displacement assay. ........................................................................... 99

Figure 4.9: Diagram as a function of continuous phase flow rate (Qoil) and capillary number (Ca) ...................................................................... 101

Figure 4.10: Droplet generation in dripping (a) and jetting (b) regimes. ............ 101

Figure 4.11: Diagram as a function of flow rate ratio (QOIL/QPEGDA) and dispersed phase flow rate (QPEGDA) operating in jetting, dripping and unstable regime ......................................................................... 102

Figure 4.12: Optical image (a) of monodisperse microparticle and relative particle size distribution (b);(c) SEM image of microparticle. .................... 103

VII

Figure 4.13: Effect of both lamp power and darocur concentration on mesh size of microparticles with PEGDA 15%. .................................................. 104

Figure 4.14: Mesh size values for different microparticle-PEGDA concentrations. ................................................................................. 105

Figure 4.15: Time lapse of fluorescence intensity (IIN/IOUT) of several probes. .. 107

Figure 4.16: Dextrans molecular weight as function of its hydrodynamic radii. 108

Figure 4.17: Dextrans partition coefficient as function of hydrodynamic radius. .............................................................................................. 109

Figure 4.18: Time lapse of fluorescence intensity (I/Imax) of several probes. ...... 109

Figure 4.19: Mechanism of Target detection. ...................................................... 111

Figure 4.20: CLSM images of the three fundamental steps of target identification: (a) T-DNA, (b) adding F-DNA, (c) displacement in presence of the Target. ........................................................................................ 113

Figure 4.21: Fluorescence intensity of (a) T-DNA, (b) adding F-DNA, (c) displacement in presence of the Target in buffer solution. ........ 113

Figure 4.22: Displacement percentage for different Target concentrations. CLSM images of microgels.. ........................................................... 114

Figure 4.23: Fluorescence intensity of (a) T-DNA, (b) adding F-DNA, (c) displacement in presence of the Target in buffer solution and (d) in human serum. ........................................................................ 114

Figure 4.24: Bead-based microfluidic assays with fluorescent microparticles ... 115

VIII

List of Tables

Table 2.1: Microgel recipes. .................................................................................. 37

Table 2.2: Characteristic parameters of microgels ................................................ 46

Table 2.3: Characteristic parameters of spherical-shell ........................................ 47

Table 2.4: Volumetric fraction calculated by both mesh size values (χξ ) and by volume values (χV). .............................................................................. 48

Table 3.1: Bulk hydrogels recipes. ........................................................................ 60

Table 3.2: Properties of bulk hydrogels of PEGDA .............................................. 64

Table 3.3: Diffusion coefficients (10-10 m2/s) of water in PEGDA hydrogels. ..... 67

Table 3.4: Diffusion coefficient of different probes in D2O and respective hydrodynamic radii. ............................................................................. 69

Table 3.5: Diffusion coefficient (10-10 m2/s) of water different probes in both water and PEGDA hydrogels. ........................................................................ 70

Table 3.6: Sequence and thermodynamic parameters of the DNA probes ........... 73

Table 4.1: Sequence, length and thermodynamic parameters of the DNA probes and RNA targets. ....................................................................................... 100

Table 4.2: Properties of PEGDA microparticles. ................................................ 105

Table 4.3: Mesh size of PEGDA (15%w/v) microparticles with different T-DNA concentrations. ................................................................................... 105

Table 4.4: Extrapolated values of τ for each probes............................................ 110

IX

List of Abbreviations

POCT Point-Of-Care Testing

PEG PolyEthylene Glycol

UV Ultra Violet

DNA Deoxyribose Nucleic Acid

RNA Ribose Nucleic Acid

miRNAs Micro RNA

PCR Polymerase Chain Reaction

qRT-PCR Quantitative Reverse Transcriptase-PCR

PDMS PolyDiMethylSiloxane

W/O Water in Oil

O/W Oil in Water

HLB Hydrophilic-Lipophilic Balance

v2 Polymer Volume Fraction

M̄c Molecular weight between crosslinks

ξ Mesh size

Qr/s Swelling ratio (relaxed/swollen state)

AFM Atomic Force Microscope

PEGDMA PolyEthylene Glycol DiMethacrylate

AAc Acrylic Acid

KPS Potassium PerSulfate

Fluo Fluoresceine O-methacrylate

PVA PolyVinyl Alcohol

Rhod Methacryloxyethyl thiocarbamoyl rhodamine B

X

DMSO Dimethyl Sulfoxide

DLS Dynamic Light Scattering

Dh Hydrodinamic Diameter

PEGDA PolyEthylene Glycol DBSAiAcrylate

NMR Nuclear Magnetic Resonance

BSA Bovine Serum Albumin

EWC Equilibrium Water Content

SR-G Sulforhodamine G

D0 Diffusion coefficient in water

D Diffusion coefficient

Rh Hydrodinamic Radius

nt Nucleotide

BHQ Black Hole Quencher

Ca Capillary Number

LMO Light Mineral Oil

SPAN 80 Sorbitan monooleate

TWEEN 20 Polyethylene glycol sorbitan monolaurate

IgG Anti-Human antibody Immunoglobulin G

CLSM Confocal Laser Scanning Microscope

SEM Scanning Electron Microscope

λex Excitation wave length

PBS Phosphate Buffered Saline

QOil/PEGDA Flow rate (continuous/dispersed phase)

df Fractal dimension

k Partition Coefficient

COUT Solute concentration of external solution

CIN Solute concentration in the gel

I Fluorescence Intensity

XI

Abstract

Biosensors technology growing attention led to the definition of specifically

customizable materials to improve the detection in complex fluids. The main focus

of this thesis has been devoted to the design of an advanced hydrogel-based tool,

properly designed for several features. The study and control of the hydrogel

structure is fundamental to achieve an accurate network that allow the transport of

selected target and work as molecular filter excluding bigger molecules and repelling

unspecific binding.

In particular, in this thesis, are described the engineering, synthesis and

characterization of PEG based hydrogel for oligonucleotide detection. Different

methods of synthesis are explored applying tagging and filtration effects. The thesis

starts with the description of synthesis and characterization of core-shell microgels

with controlled structural properties. Core-shell PEG-based microgels, with a size

ranging between 0.5 and 1.3 μm, were obtained by combining precipitation and

seeded polymerizations. We demonstrated the possibility of tailoring and controlling

the bulk and surface properties. Then a structural characterization was attained

calculating polymer volumes fraction from AFM images and combined these with

equilibrium swelling theory in order to determine the mesh size of the microgels.

In such away, we were able to describe the organizations of the different adlayers,

highlighting the possibility of some overlap of the adlayers representing physical

barriers at the boundaries of each shell. These multifunctional microgels were used

in multiplex assays for their favorable capability to accommodate encoding systems

and anchoring groups for probes to capture circulating targets.

In the second part of the work, we described PEGDA bulk hydrogels, synthesized by

UV radical photopolymerization and characterized by their swelling parameters.

XII

Results show a mesh size values between 2.6-1.6 nm. Moreover, diffusion studies of

several probes with different hydrodynamic radii were accomplished using NMR

technique. Results showed that diffusion of small molecules, as sulforhodamine G

was not affected by decrease of mesh size values, which, by the contrast, slow down

the diffusion of bigger molecules as dextrans. All these characterizations were

preliminarily done on bulk hydrogels in order to customize the network to obtain a

specific structure and desired final properties. Then same recipes were scaled-down

to synthetize microparticles by microfluidic device improving the shape and size

control and to reduce both production time and costs. Microparticles so obtained

were characterized by swelling parameters showing the same trend of ξ obtained for

bulk. Finally, we focused on the probe design to achieve our customized platform

for biomarkers detection, in particular for miRNA143-3p which play an important

role in the function and formation of the cardiac chamber. Our double strand probe

consisted of a short methacrylate DNA-Tail, covalently bound to the polymer

networks, and a longer fluorescent DNA sequence. The latter was partially

complementary to DNA-Tail and totally complementary to a specific target. Target

identification occurred by double strand displacement assay. Using this probe

scheme, we included, directly in flow, the capture element of the target to accomplish

functionalized particles. Then were characterized in term of their swelling behavior,

using confocal light microscopy. Moreover, the CLSM images allowed to evaluate

the fluorescence intensity of our hydrogels for each step to determine displacement

percentage and efficiency of our detection system. Results showed a 93% of

displacement proving the good efficiency of our detection system in both buffer

solution and in complex fluids such as the human serum.

To sum up, in this thesis is shown the importance of the choice of materials and their

characterization to modulate and predict final properties. In this way, it is possible to

synthetize materials with specific features and capture elements to combine both

molecular filter capability and target detection. These engineered hydrogels

represent in fact a tunable, fast, costless and easy platform for biomarkers detection.

1

Chapter 1

Introduction

In biosensor field, no-wash assay have been found to be particularly attractive due

to their extraordinary potentiality. In fact, these analytical devices represent a new

generation assay, easily performed mixing the signal generator probes with the

sample solution. Compared with conventional biosensor, the highest advantages of

no-wash assay are correlated to their user-friendly and time saving properties,

portability and low costs. All these features make them suitable for point-of-care

testing (POCT) development.

These biosensors can be developed using different materials and, among these,

polymers are the common choice. In particular, hydrogels are suitable for this

purpose due to their tunable chemistry and possibility to modulate their network

structure.

Therefore, in this work we show the possibility to synthesize functionalized

hydrogels with a flexible chemistry, tunable control of the network and specific

capture molecules. In such a way, we are able to accomplish a bead-based assay

merging both molecular filter and detection capabilities (Figure 1.1).

Figure 1.1: Hydrogels with (a) tunable control of mesh size (b) versatile chemistry to allow the conjugation of capture molecule in different position; and (c) molecular filter capability, with

antifouling properties and specific capture of a given target.

2

1.1 Hydrogels: principal concepts

Hydrogels are a class of polymer networks which, due to their hydrophilic nature,

may absorb large amount of water1 and physiological fluids while remaining

insoluble in aqueous solutions. The large water content lends them a high flexibility

comparable with natural tissue. Due to their good ability in wide range of

applications, they are widely studied for the past 50 years2-3 and there is an

increasingly interest of the scientific community on the hydrogel topic (Figure 1.2).

Figure 1.2: Histogram showing the increase in publications related to the keyword “hydrogel” during the past 50 years. PubMed data.

In particular, they can be used in several biomedical applications including

diagnostic,4-5 drug delivery,6-7 tissue engineering8-9 and pharmaceutical

applications.10 The structure of a hydrogel is usually an elastic and porous mesh

where biological molecules can be incorporated or entrapped within. These networks

are composed of homopolymers or copolymers, based on the method of preparation.

Modifying the density of the network, the mechanical properties of the material and

their mesh can be properly modulated to suit specific applications.

Hydrogels are generally characterized by several parameters such as the final

capability to absorb liquids (swelling thermodynamics), the rate at which the liquid

3

is absorbed into their structure (swelling kinetics), as well as their mechanical

property in wet or hydrated state (wet strength).

Chemical functionalization of this polymeric hydrogels is widely studied with

the aim of adding on the surface or in the network, several molecules as peptide,

enzyme or oligonucleotide to allow the detection of different target.11-13

1.2 Hydrogels for biosensing

As defined 1999 by the IUPAC, “a biosensor is a self-contained integrated device

which is capable of providing specific quantitative or semi-quantitative analytical

information using a biological recognition element (biochemical receptor) which is

in direct spatial contact with a transducer element. A biosensor should be clearly

distinguished from a bioanalytical system, which requires additional processing

steps, such as reagent addition. Furthermore, a biosensor should be distinguished

from a bioprobe which is either disposable after one measurement, i.e. single use,

or unable to continuously monitor the analyte concentration” (Figure 1.3).14

Figure 1.3: Schematic illustration of biosensor.

The first biosensor was described in 1962 by Clark and Lyons who immobilized

glucose oxidase (GOD) on an amperometric oxygen electrode surface in order to

quantify directly the glucose concentration in a sample.15-16

4

Biosensors represent an advanced platforms for biomarker analysis with

the advantages of being easy to use, inexpensive and rapid as well as offering

multi-analyte testing capability.17

Hydrogels for biosensor can be prepared in aqueous solutions by different synthesis

procedure as thermo-initiated18 or UV19 radical polymerization. Moreover, adding

recognition motifs such as peptides, enzyme or oligonucleotides, is possible to use

hydrogels for detection applications. Polymer network and hydrophilic environment

allow the diffusion of different molecules through the hydrogel matrix. However,

diffusion of larger analytes, such as proteins, can be prevented by an increasing

crosslinking density and an appropriate choice of materials.20

These biosensors can be developed using different materials. In particular, polymers

are widely used and, among these, polyethylene glycol (PEG) hydrogels results to

be the suitable monomer to attain low-fouling properties, enhancing the specificity

of the capture elements. Moreover, PEG derivatives (Figure 1.4) are extensively

adopted for their good solubility in aqueous solution, low-cost production at different

molecular weights and chemical functionalities.21

Figure 1.4: Chemical structures of PEG and its di(meth)acrylate.

These materials are usually prepared using the free-radical polymerization of

reactive methacrylate PEG derivatives or polyethylene glycol di-acrylates (PEGDA)

in the presence of a UV or thermal initiator. The light or temperature induce the

5

photoinitiator activation generating a benzoyl free radical through a homolytic

scission of a C–C bond, subsequently triggering the covalent crosslinking of the gel.

1.3 Biomarkers detection

Biomarkers cover a broad range of biochemical entities, such as proteins, nucleic acids,

small metabolites, sugars and tumor cells found in the body fluid (Figure 1.5).

They are widely used for risk assessment, diagnosis, prognosis, and for the prediction

of treatment efficacy and toxicity.22-23

Figure 1.5: Most common biomarkers.

In particular, nucleic acids (DNA and RNA), due to their high specificity, can be

amplified to increase abundance in most applications, and can be labelled

(for detection) using different approaches, resulting extremely attractive targets for

diagnostics. The assessment of both nucleic acid sequence and relative expression

level is extremely important for diagnostics applications. In fact, both mutations

(changes in nucleic acid sequence) and abundance of nucleic acid target can indicate

disease. Their up- or down- regulation is an important indicator for applications like

6

drug discovery and cancer diagnostics.24 In biomarkers context, miRNAs have

attracted high attention due to their immense regulatory power so that the variation

of their levels can be utilized as potential biomarkers for the diagnosis and prognosis

of a variety of diseases.25-29 MicroRNAs (miRNAs) are a class of small non-coding,

highly conserved, single stranded RNAs. According to the current database

(http://www.mirbase.org), there are total 1881 annotated human miRNA precursor

genes that processes 2588 mature miRNAs. Although miRNAs abound in tissues,

it has been shown that there are also circulating miRNAs in body fluids such as

plasma, urine, saliva, seminal fluid, amniotic fluid, breast milk, bronchial lavage and

cerebrospinal fluid.30 Mature miRNAs are approximately 21 nucleotides in length,

with a good stability in a wide range of biological contexts, and play important roles

in the development and cellular process with regulatory tasks on the genome

expression.31-33 In addition to these vital processes, miRNAs are implicated in

diverse cellular activities, such as immune response,34 insulin secretion,

neurotransmitter synthesis, circadian rhythm,35 and viral replication. Recent

genome-wide analyses have also identified deregulated miRNA expression in human

malignancies. MiRNAs can modulate oncogenic or tumor suppressor pathways,

whereas miRNAs themselves can be regulated by oncogenes or tumor suppressors.36

MiRNAs have been reported to associate with a number of pathological conditions

of the central nervous system, such as Alzheimer’s and Parkinson’s.37 In addition,

the roles of miRNAs in the pathological processes of heart, vascular tissue, and blood

have been popularly studied. Among these, several researches are focused on the role

of miRNAs in cardiovascular pathologies38-39 such as arrhythmias,40 fibrosis,41

pressure overload-induced remodeling,42 and metabolic disorders.43 Correlation

between disease state and expression level of these circulating species suggest that

miRNAs can be strong candidates for the development of non-invasive biomarker

screens, for the early and asymptomatic detection of disease and for the monitoring

of the treatment response.44

7

Extracellular miRNAs circulating in the blood of both healthy and diseased people

showed extreme stability, and resistance to degradation from RNases activity.

Most of the circulating miRNAs are well protected from RNases because they can

reside in membrane structure of microvesicles such as exosomes, microparticles, and

apoptotic bodies.29

Figure 1.6: Compartmentalization of circulating miRNAs. Circulating miRNAs are contained within vesicles, in protein complexes, and in lipoprotein

complexes. Adapted with permission form Ref [29] Copyright 2013, American Chemical Society.

In any given diagnostic test, the targets must be manipulated, captured, or detected.

For a test to be meaningful, it must be specific for the target of interest and sensitive

enough to detect entities at physiologically relevant quantities. For this purpose,

nucleic acids results an ideal choice due to their intrinsic molecular base pairing

ability, offering a high degree of selectivity and stability. Oligonucleotide detection

is based on specific hybridization between a probe that is a single-stranded nucleic

acid oligonucleotide and the target, the sequence to be detected.

Well-known conventional technologies for the detection of oligonucleotides, based

on hybridization to complementary probes, are represented by microarray

analysis,45-46 polymerase chain reaction (PCR, qRT-PCR)47 and oligonucleotide

probes such as molecular beacons48 and double strands (Figure 1.7).49

8

However, for all these techniques, extraction, amplification, and calibration steps are

needed resulting time-consuming. Moreover, preliminary amplification step may

compromise the assay accuracy.17, 30, 47

Figure 1.7: Conventional techniques for miRNAs detection.

Novel suspension arrays offer several advantages such as the high flexibility

(obtained by simply changing/adding probe particles), enhanced reaction kinetics

due to radial diffusion, lower costs and sample consumption and shorter

incubation.50-54 Suspension arrays involving polystyrene beads doped with

combinations of dyes for optical coding have been carried out by Luminex.55 In order

to improve the multiplexed readout, suspension array technologies with barcodes,

silica nanotubes, Au/Ag nano-barcodes, and dot-coded polymer particles have been

developed.53-55 Then, particle-based suspension arrays have been attracting

increasing interest for the multiplexed detection of nucleic acids, offering high

flexibility, easy probe-set modification, and high degrees of reproducibility.56

9

A direct and absolute quantification of circulating miRNAs in body fluids is

particularly needed even though challenges are represented by their small size, high

level of sequences homology, demand of high sensitivity and specificity.

1.4 Classifications of hydrogels

The classification of hydrogels depends on many factors such as their physical

properties, nature of swelling, method of preparation, ionic charges, and nature

of crosslinking.57 A first classification can be based on gel dimensions.

Typically, hydrogels can be categorized as microgels, microparticles and macrogels.

The first ones are defined as colloidal stable system, with a water swellable

polymeric networks whose diameter typically ranges from 100 nm to 1 μm.

Hydrogels microparticles, instead, show a range from tens to hundreds of microns

and can be considered a sort of macrogels. These are bulk, monolithic networks

with a typical range in size from hundreds of microns or greater.

Microgels are physically different from macrogels even though internally have the

same gel structure. Indeed, as the high surface to volume ratios are several orders of

magnitude larger than those in macrogels, microgels can present a non-uniform

distribution of polymer chains throughout the network.58-59 The synthesis of

microgels particles typically involves a nucleation, aggregation and growth

mechanism that ultimately results in a non-uniform distribution of polymer chains

throughout the network.60-61

Hydrogels can be also classified into different categories based on their crosslinking

chemistry.62 The first one involves physical gels, which are defined as polymeric

networks that are bound together via polymer chain entanglement and/or

non-covalent interactions that exist between polymer chains.1, 63-65 The attractive

forces holding these networks together are typically based on hydrogen bonding,

electrostatic or hydrophobic interactions and thus, the gels can be reversibly

10

dissolved under certain conditions that would weaken these attractive forces,

i.e. a change in pH. In contrast to these weak physically crosslinked networks,

the other general class of hydrogels consist of the chemical crosslinked gels.

These hydrogels exhibit improved stability due to the formation of covalent bonds

between different polymer chains throughout the networks and display endurance

with respect to network structure.1, 66-67 These gels are usually formed through

monomer polymerization in the presence of a crosslinking agent, which is typically

a monomer with at least two polymerizable functional moieties.

Moreover, hydrogels can be also categorized as neutral or ionic, based on the nature

of side groups. In neutral ones, the driving force for swelling is due to the water-

polymer thermodynamic mixing contribution to the overall free energy, along with

elastic polymer contribution.10 The swelling of ionic hydrogels is also affected by

the ionic interactions between charged polymers and free ions.68 This kind of gel,

containing ionic groups (such as carboxylic acid) can imbibe larger amount of water

because of its increased hydrophilicity.

Finally, hydrogel can also be grouped in terms of their interaction with the

surrounding environment. Non-responsive gels are simple polymeric networks that

dramatically swell upon exposure to water. Depending on its structure, hydrogel can

respond to environmental changes by changing its size or shape. Most important

factors that trigger a hydrogel response are temperature,64, 69-70 pH,71-72 ionic

strength,73-74 light,63, 75-76 and electric field77 (Figure 1.8).

Figure 1.8: Principal environmental factors affecting hydrogels size.

11

As far as the temperature is concerned, hydrogels containing hydrophobic groups, or

those susceptible to chain aggregation, respond to the temperature change with a

great extent. Because of both solubility and swelling are driven by the same forces,

the response of the hydrogel to temperature change can be either direct or inverse.

The solubility and swelling of the hydrogel can increase with increase in temperature,

while an opposite trend is observed with inverse thermo-responsive hydrogels.

Hydrogels can also change their size with the change in the composition of the

swelling medium. The hydrogel response is deeply affected by the presence of salt

and non-solvent in the swelling solution.

Because hydrogels are tunable, responsive, and chemically versatile, they are

suitable platform in the design of many biomaterials,78 where the properties of the

polymer may be engineered to suit specific medical applications.

1.5 Hydrogels: synthesis procedure

Hydrogels may be synthesized in a several ‘‘classical’’ chemical ways.

These include one-step procedures like polymerization and parallel crosslinking of

multifunctional monomers, as well as multiple step procedures involving synthesis

of polymer molecules with reactive groups and their subsequent crosslinking.

One of the most common technique is synthesis in batch.79 This procedure is usually

radical photo or thermal-initiated by using appropriate initiators or, for complex

architecture, the synthesis can be performed via living controlled radical

polymerizations. It is possible to identify three different preparation strategies, based

on the particle formation mechanism: homogeneous nucleation, emulsification and

complexation. In the first one microgels are generated from an initially homogeneous

solutions. On the other hand, during the emulsification, aqueous droplets of a pre-

gel solution are formed in an oil phase and, then, the droplets are polymerized

becoming microgels. Finally, microgels can be synthesized by mixing two dilute

12

water-soluble polymers that form complexes in water. The new generation of

polymer materials are designed and synthesized with an high control of the structure

(crosslinking density) and with tailored properties such as swelling behavior and

chemical and biological response to stimuli.80-81

In order to obtain a good control on the size, shape and chemistry, hydrogels can be

produced using miniaturized devices such as microfluidic. In fact the recent years,

advance of microfabrication methods, allow to synthesize tunable microparticles

with a significant reduction in terms of time, costs and reagent needed.82

Microfluidic assisted methods are usually based on formation of a stable emulsion

of an oil phase (as continuous phase) and a water phase constituted by a mix of

monomers and biomolecules. The synthesis occurs in a miniaturized devices

made in glass or polydimethylsiloxane (PDMS).83 The crosslinking of polymeric

monomers is performed by shining UV/Vis light on the flowing liquids in-situ or

in specialized compartments activating a suitable photoinitiator based on

hydroxyalkylphenone species (i.e., Darocur, Irgacure). Benzoyl free radical is

produced upon the homolytic scission of C–C bonds in the photosensitive

molecules that allows the cross-links formation in the gel.84 The intensity of light,

the exposure time and the concentration of photoinitiator are determinant on the

double bond conversion and in turn on the mechanical rigidity and the pores of

the gels. Microfluidic techniques can be divided in droplet generation based

methods and flow lithography (Figure 1.9). Droplet forming micro-devices have

been described since 2005 as continuous on-chip production of water-in-oil

emulsions based on the break-off of droplets in two-phases at T-junction or in

flow-focusing geometries.85-86 An innovative approach has been developed by

Doyle et al.11, 87 that reported an innovative method for the continuous production

of hydrogel particles under flow in a PDMS microfluidic device with high

throughput. With this technique, they were able to obtaining a high number codes

by combining the graphical and spectral encoding.87-88 This approach has been

defined as continuous flow lithography and it relies on rectangular microfluidic

13

channel where multiple co-flowing streams pass over a pulsing UV light, that is

projected through a mask from the objective, to give complex shapes.

Multiple shapes and sizes have been obtained as well as differential chemistries,

such as Janus particles, are possible. An evolution of this technology is

represented by stop-flow lithography where the pumping system is actuated so

that the flow is stopped for few milliseconds allowing the polymerization through

a mask. In such a way Doyle’s group obtained particles with graphical and

spectral encoding as well as with multiple capture probes positioned in different

regions.89

Figure 1.9: Different microfluidic techniques: (a) Flow focusing device for functionalized microparticles; (b) Droplet microfluidics for functional Janus microparticles production;

(c) Continuous flow lithography for complex shaped hydrogel microparticles. Respectively adapted with permission from : Ref.[90] Copyright 2016, Elsevier B.V.; Ref. [91] Copyright 2011, Royal Society of Chemistry; Ref. [89] Copyright 2014, Rights Managed by Nature Publishing Group.

14

Regarding polymerization techniques, exist several kind of strategies, such as bulk

polymerization, solution polymerization/crosslinking and suspension

polymerization or inverse-suspension polymerization. Polymerized hydrogels may

be produced in a wide variety of forms including films and membranes, rods,

particles, and emulsions. High rate of polymerization and degree of polymerization

occur because of the high concentration of monomer. The bulk polymerization of

monomers, to make a homogeneous hydrogel, produces a glassy and transparent

polymer matrix, which is very hard. When immersed in water, the glassy matrix

swells to become soft and flexible. In solution copolymerization/crosslinking

reactions, monomers are mixed with the multifunctional crosslinking agent.

The polymerization starts by UV-irradiation or by a redox initiator system.

Hydrogels so obtained need to be washed to remove the monomers, crosslinking

agent, the initiator, and other impurities. Phase separation occurs and the

heterogeneous hydrogel is formed when the amount of water during polymerization

is more than the water content corresponding to the equilibrium swelling. Typical

solvents used for solution polymerization of hydrogels include water, ethanol,

water–ethanol mixtures, and benzyl alcohol. Then the synthesis solvent may be

removed after formation of the gel by swelling the hydrogels in water. Suspension

polymerization is an advantageous method since the products are obtained as

microspheres and thus, grinding is not required. Since water-in-oil (W/O) process is

chosen instead of the more common oil-in-water (O/W), the polymerization is

referred to as ‘‘inverse-suspension’’. In this procedure, the monomers and initiator

are dispersed as homogenous mixture. The viscosity of the monomer solution,

agitation speed, rotor design, and dispersant type mainly governs the resin particle

size and shape. The dispersion is thermodynamically unstable and requires both

continuous agitation and addition of a low hydrophilic–lipophilic-balance (HLB)

suspending agent.

15

1.6 Chemical an physical hydrogel characterization

In this section, we report on the chemical and physical properties of hydrogels that

affect primarily their behavior. The three-dimensional and potentially responsive

nature of the polymer networks plays a key role in bead-based assays and, when

considering hydrogel materials in biosensing applications, a number of concerns has

to be take into account. Particularly important are the issues related to the capability

of capturing the given target and to the mass transport inside the network closely

connected to the swelling behavior and diffusion of solutes.

1.6.1 Swelling behavior

Hydrogels are crosslinked polymer networks swollen in a liquid medium. They are

able to absorb from 10-20% up to thousands of times their dry weight in water.

When a dry hydrogel begins to absorb water, the first water molecules entering the

matrix will hydrate the most polar, hydrophilic groups, leading to primary bound

water. Once the polar groups are hydrated, the network swells, and exposes

hydrophobic groups, which also interact with water molecules, leading to

hydrophobically-bound water, or secondary bound water. Primary and secondary

bound water are often grouped in the so-called total bound water.

After the polar and hydrophobic sites have interacted with and bound water

molecules, the network will imbibe additional water, due to the osmotic driving force

of the network chains towards infinite dilution. This additional swelling is hindered

by the covalent or physical crosslink, leading to an elastic network retraction force.

Thus, the hydrogel will reach an equilibrium swelling level. The additional swelling

water that is imbibed after the ionic, polar and hydrophobic groups become saturated

with bound water is called free water or bulk water and is assumed to fill the space

between the network chains, and-or the center of larger pores, macropores or

voids.1, 92 The hydrogels biocompatibility and crosslinked structure are responsible

for their different applications. The nature of monomers, method of preparation,

16

and nature of crosslinking agent determine the crosslinked structure of the gel.

This can be characterized studying the gel swelling behavior. Several theories have

been proposed to explain the network structure of the gel, as well as the mechanism

of their swelling. Some theories examine the real network structure with its defects,

while others, for analysis simplicity, consider the network as an ideal structure.

For biomedical purposes, the hydrogel network is considered ideal.

The most important parameters used to characterize network structure are the

polymer volume fraction ( 2), molecular weight of the polymer chain between two

neighboring crosslinks (M̄c ), and the corresponding mesh size (ξ).10 These three

parameters, which are correlated to one another, describes the nanostructure of the

crosslinked hydrogels and can be determined theoretically or using many

experimental techniques. In particular two prominent theory are frequently used to

elucidate the structure of hydrogels: equilibrium-swelling theory93 and rubber-

elasticity (Figure 1.10).94

Figure 1.10: A crosslinked hydrogel structure with the main swelling parameters:

mesh size (ξ) and the average molecular weight between crosslinks( M̄c ). Reproduced with permission from Ref.[20], Copyright 2012 Elsevier Ltd.

17

1.6.2 Equilibrium swelling theory

The structure of hydrogels that do not contain ionic moieties, can be analyzed by the

Flory–Rehner theory.95 It states that a crosslinked polymer gel, that is immersed in a

fluid and allowed to reach equilibrium with its surroundings, is subject only to two

opposing forces: the thermodynamic force of mixing and the retroactive force of the

polymer chains. At equilibrium, these two forces are equal. This physical situation

is defined in terms of the Gibbs free energy (Equation 1.1).

ΔG = ΔG + ΔG Equation 1.1

Here, ΔGelastic is the contribution due to the elastic retroactive forces developed inside

the gel, and ΔGmixing is the result of the spontaneous mixing of the fluid molecules

with the polymer chains. The term ΔGmixing is a measure of the compatibility of the

polymer with the molecules of the surrounding fluid. This compatibility is usually

expressed by the polymer-solvent interaction parameter, χ. Differentiation of

Equation 1.1 with respect to the number of solvent molecules while keeping

temperature and pressure constant results in Equation 1.2:

μ − μ , = Δμ + Δμ Equation 1.2

Where μ1 is the chemical potential of the solvent in the polymer gel and μ1,0 is the

chemical potential of the pure solvent. At equilibrium, the difference between the

chemical potentials of the solvent outside and inside the gel must be zero. Therefore,

changes in the chemical potential due to mixing and elastic forces must balance each

other. The change of chemical potential due to mixing can be expressed using heat

and the entropy of mixing. By the contrast, the change in chemical potential due to

the elastic retroactive forces of the polymer chains can be determined from the theory

of rubber elasticity.96-97 Upon equaling these two contributions, an expression for

determining the molecular weight between two adjacent crosslinks of a neutral

hydrogel prepared in the absence of a solvent can be obtained.10, 98-100 The molecular

18

weight between two consecutive crosslinks, which can be either chemical or physical

in nature, is a measure of the degree of crosslinking of the polymer and can be related

to physical properties including swelling behavior and stiffness. It is important to

note that due to the random nature of the polymerization process itself only average

values of M̄c can be calculated Equation 1.3

= − , , ,,/ , Equation 1.3

In this equation M̄n is the molecular weight of the polymer chains prepared in the

absence of a crosslinking agent, v2,s is the polymer fraction in the swollen state, χ1,2

is the polymer-solvent interaction parameter, ̄ν is the specific volume of the polymer,

and V1 is the molar volume of water.

In particular, polymer volume fraction in the swollen state characterizes how well

the polymer absorbs water. It can be expressed as the ratio of the volume of dry

polymer, Vd, to the volume of the swollen polymer gel, Vs, and is the reciprocal of

the degree of swelling, Q.101 The polymer fraction in the swollen state can easily

be determined with equilibrium swelling and lyophilization experiments using

Equation 1.4 above.

v , = = Equation 1.4

If the volume of the polymer and solute can be assumed additive, Equation 1.5 can

be considered:

V = V + V Equation 1.5

Then the dry and swollen mass of the gel can be used to find the polymer volume

fraction in the swollen state, applying Equation 1.6, where ρsolvent is the density of

19

the solvent, ρpolymer is the density of the polymer, mswollen is the mass of the swollen

hydrogel, and mdry is the mass of the dry hydrogel after lyophilization.

v , = Equation 1.6

Regarding molecular weight between crosslink, Peppas and Merrill102 modified the

original Flory-Rehner theory for hydrogels prepared in the presence of the water.

The presence of the water effectively modifies the change of chemical potential due

to the elastic forces. Equation 1.7 predict the molecular weight between crosslinks

in a neutral hydrogel prepared in the presence of water; v2,r is the polymer volume

fraction in the relaxed state, which is defined as the state of the polymer after

crosslinking, but before swelling.

= − , , ,, ,

,⁄ ,

, Equation 1.7

The presence of ionic moieties in hydrogels makes the theoretical treatment of

swelling much more complex. In addition to the contributions of ΔGmixing and

ΔGelastic in Equation 1.1, there is an additional contribution to the total change in

Gibbs free energy due to the ionic nature of the polymer network, ΔGionic

(Equation 1.8):

ΔG = ΔG + ΔG + ΔG Equation 1.8

Upon differentiating Equation 1.8 with respect to the number of moles of solvent,

keeping T and P constant, an expression similar to Equation 1.2 for the chemical

potential can be derived (Equation 1.9):

μ − μ , = Δμ + Δμ + Δμ Equation 1.9

20

Here, the Δμionic is the change in chemical potential due to the ionic character of the

hydrogel.

1.6.2.1 Rubber elasticity theory

Hydrogels resemble natural rubbers in their remarkable property to elastically

respond to applied stresses. A hydrogel subjected to a relatively small deformation

(less than 20%) will fully recover to its original dimension rapidly. This elastic

behavior of hydrogels can be used to explain their structure applying the rubber-

elasticity theory originally developed by Treloar97 and Flory94 for vulcanized rubbers

and then modified by Flory for polymers.96 An expression (Equation 1.10) has also

been developed for relating the molecular weight between crosslinks and the elastic,

or Young’s, modulus E,

= Equation 1.10

The relationship should be taken as a rough estimation which often underestimates

M̄c since it does not take into account physical crosslinks or chain ends.103

Another important parameter used to describe hydrogels is the mesh size, or

characteristic length, ξ, which represent the characteristic distance between

crosslinks, provides a measure of the space available between the

macromolecular chains and it is an important factor in solute transport in

hydrogels. The mesh size also affects properties such as mechanical strength,

degradability, and diffusivity within a hydrogel. If a solute molecule is larger than

the mesh size, it is theoretically impossible for the solute molecule to move

through the hydrogel. The mesh size of a hydrogel is affected by the degree of

crosslinking, chemical structure of the monomer, and other external stimuli, such

as pH, temperature, and the presence of ions. Again, it can be reported only as an

average value. The mesh size can be related to other parameters discussed above

21

via scaling relationships, which produce the Equation 1.11, where r̅ ⁄ is the

root-mean-squared, unperturbed, end to end distance of the polymer chain

between two adjacent crosslinks.

ξ = v , ⁄ r ⁄ Equation 1.11

This statistical parameter can be found via a relationship first developed by Flory,

Equation 1.12:

r ⁄ = CnNl2 Equation 1.12

where Cn is the Flory characteristic ratio, l is the length of each link and N, the

number of links in the chain, is given by Equation 1.13:

N = Equation 1.13

Where Mr is the molecular weight of the repeating unit of the polymer.

1.6.2.2 Factors affecting swelling of hydrogels

One of the most important factors that affects the swelling of hydrogels is the

crosslinking ratio. It is defined as the ratio of the moles of crosslinking agent to the

moles of polymer repeating units. The presence of crosslinks represent a hindrance

to the mobility of the polymer chain.10 In fact, a high value of crosslinking ratio is

associated with a tighter structure that consequently will swell less compared to the

same hydrogels with lower crosslinking ratio. Moreover, the chemical structure of

the polymer may also affect the swelling ratio. For example, hydrogels containing

hydrophilic groups swell more than hydrogels containing hydrophobic one. The

latter, in fact, collapse in the presence of water in order to minimize their exposure

to the water molecules.10 Finally, as described previously, there are environmentally

22

sensitive-hydrogels. For these materials, there are many specific stimuli, as pH,

temperature, light, that can affect their swelling.

1.6.3 Solute diffusion within hydrogels

The diffusion of solutes in hydrogels is widely studied in several fields.

Hydrogels are in fact used in separation processes such as chromatography,104 for

the encapsulation of cells for both biomedical and fermentation purposes, in

biosensors and as biomaterials for the delivery of drug and prosthetic

applications.105-106

The one feature of hydrogels that all these applications capitalize upon is their

ability to restrict the diffusive movement of a solute. Solute transport within

hydrogels occurs primarily within the water-filled regions in the space delineated

by the polymer chains.

Any factor, which reduces the size of these spaces, will have an effect on the

movement of the solute. Several factors such as the size of the solute related with

the size of the openings between polymer chains, the fractal dimension of the

diffusing species, polymer chain mobility and the existence of charged groups on

the polymer, might influence the solute mobility.

In particular, the polymer chain mobility is an important factor governing solute

movement within the hydrogel. In general, the diffusivity of a solute through a

physically crosslinked hydrogel decreases as crosslinking density increases, as

the size of the solute increases and as the volume fraction of water within the gel

decreases.107 The fractal dimension of the diffusing species df is dependent from

rigidity of the molecule, in particular for a rigid sphere, df =3.0, for a linear

Gaussian polymer coil (random walk) df =2.0 and df < 2 for polymers with

excluded-volume interactions.108

23

Figure 1.11: Parameters that influence diffusion of probe particles in polymeric media. Adapted with permission from Ref.[108], Copyright 2002, American Chemical Society.

For all these reasons, the understanding of the parameters governing solute diffusion

within hydrogels as well as the means by which they affect diffusion are important

to be studied. Therefore, a number of mathematical expressions have been developed

in an effort to model solute diffusion in hydrogels.109

1.7 Aim and outline of dissertation

The growing interest in biosensor field has driven us to study novel materials, to

improve the detection in complex fluids.

Although different types of microparticles are already on the market

(e.g., fluorescent labeled and magnetic), there is a need for materials with high

flexibility to introduce the right functionalities for the encoding and for the capture

elements with enhanced properties. A direct and absolute quantification of

circulating miRNAs in body fluids is challenging, due to the high level of sequences

homology, small size and demand of high sensitivity and specificity.

24

In order achieve this, we focused on the research, synthesis and optimization of

materials, which combine features of biocompatibility, stability and low interaction

with proteins. This work is, in fact, focused on the challenge to combine these

properties with flexible chemistry and tunable control of the network.

Therefore, we synthesized engineered PEG-based materials, properly functionalized,

with versatile chemistry and tunable control of mesh size allow to obtain a

customized molecular filter for a simple detection of oligonucleotides in complex

fluids. Our particle-based assay allow to overcome limitations of conventional

techniques, previously described. This kind of assay is based on optical detection

scheme for oligonucleotide. Capture element can be added on the hydrogel surface

or inside in order to achieve smart materials for specific detection.

In Chapter 2, we focused on the synthesis and characterization of core-shell

PEGMA-microgels, obtained by combining precipitation and seeded

polymerizations, with controlled structural properties. AFM images, combined with

equilibrium swelling theory, allowed to describe the organizations of the different

adlayers highlighting the possibility of some overlap representing physical barriers

at the boundaries of each shell.

In Chapter 3, synthesis and characterization of PEGDA-bulk hydrogels, properly

functionalized with capture element, were done. Swelling studies, combined with

both NMR and fluorimetric analysis for probes diffusion, allow to optimize the

network structure to combine both molecular filter and capture element capabilities.

Finally, in Chapter 4, this knowledge were scaled down to synthetize microparticles

by microfluidic device improving the shape and size control and to reduce both

production time and costs. Moreover, adding directly in flow a probe, properly

designed for the detection of miRNA143-3p (responsible of cardiovascular diseases)

we were able to detect, also in complex fluids the presence of the interest target.

Conclusions and future perspectives are synthetically presented and discussed in

Chapter 5.

25

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33

Chapter 2

Core-shell microgels:

synthesis and swelling characterization

Abstract. Due to their versatility and their modulating properties, microgels have been largely studied in biomedical field for their wide application in diagnostic. In particular, multifunctional core-shell microgels, with complex architectures, have been used for multiplex assays. Changing synthesis parameters was possible to accommodate encoding system and anchoring group for specific probes in order to capture circulating targets. In this chapter, we focused on the synthesis and characterization of a fluorescent encoded poly(ethyleneglycol)-based microgels with a core-shell architecture obtained combining precipitation and seeded polymerizations. Structural characterization was done combining AFM images with equilibrium swelling theory. In such a way, we were able to describe the organizations of each single shell, evaluating the presence and effects of the overlaps as physical barriers.

The work described in this chapter is part of a manuscript published: E. Battista, A. Mazzarotta, F. Causa, A.M. Cusano, P. A. Netti. “Core−shell microgels with controlled structural properties”. Polymer International, (2016). 65(7), 747-755.1 Part of this work is reproduced or adapted with permission from [1], Copyright 2016 Wiley.

34

2.1 Introduction

Hydrogels are three-dimensional, and potentially responsive, polymer networks

that, due to their customizable physical and chemical properties, has been widely

used in biomedical field. In particular, recent studies have focused on

multifunctional hydrogel particles as materials platforms to attain the new

generation of diagnostic tools.2-5 Multifunctional microgels require that the

chemistries of the surface and the bulk could be independently modified to

include the appropriate molecules for the desired purpose. The flexible chemistry,

in fact, allow to functionalize these materials with several capture elements able

for the detection of different targets as biomarkers6-7 or pollutants.8

In the diagnostic field, microgels have been extensively applied in bead-based

multiplex assays, an evolution of traditional immunoassays. For this purpose,

the recognition motif was directly mounted on the surface of encoded microgels

to test panels of targets in a single reaction vessel.2

Conventional technique for the synthesis of microgels, based on a single-step

polymerization, involves a nucleation, aggregation and growth mechanism that

ultimately results in a non-uniform distribution of polymer chains throughout the

network. Therefore, new strategies has been studied to attain a high control of the

particle size distribution, the colloidal stability, and the distribution of specific

functional groups.

To do this, core-shell architecture by two-step “seed–feed” polymerization were

synthesized.7 The core-shell architecture resulted was very interesting having

hydrogel compartmentalization with differentiated proprieties in both core and

the shells and almost all kinds of functional groups could be incorporated into

microgel particles by one-pot copolymerization, two-step “seed-feed”

polymerization, and post-functionalization.9 Moreover, changing the recipe, is

possible to achieve different phase-transition behavior with temperature or pH.

35

Several techniques have been used so far to investigate the structural and

chemical properties of microgels either directed to the bulk and of surface.

On the other hand, not enough studies has been already done for the study of

swelling parameters of microgels.

The traditional technique used for macrogels structural characterization is based

on the equilibrium swelling theory developed by Flory and Rehner10 and then

modified by Peppas and Merrill.11 This theory has not been frequently applied to

microgels, as difficulties arisen in the measurements of swelling of such

micro-objects in the canonical way. Peppas et al. developed a method for the

direct determination of mesh size and molecular weight between crosslinks of

microgels by applying an equilibrium swelling theory through atomic force

microscopy (AFM).

In particular, in their work, particle volume size was determined by average

diameter of spherical microgel measured by AFM.11 Possible deformations due

to collapse of microgels on the surface were not considered. In order to overcome

the mistake associated with this possible collapse, we propose in this chapter an

innovative swelling characterization obtaining by the measure of the volume of

each particle directly form AFM images by “Laplacian volume”.

For this characterization, multifunctional microgels with core-shell architecture

were synthesized and provided by a wide range of fluorescence-based codes.

In particular core double shell microgels, made of poly(ethyleneglycol)

dimethacrylate (PEGDMA), were obtained by copolymerization of two different

dyes and acrylic acid, all positioned in a spatially controlled manner with the aim

to accomplish an encoding system and provide carboxyls as anchoring groups for

subsequent conjugations.

36

2.2 Experimental section

2.2.1 Materials

Poly(ethylene glycol) dimethacrylate (PEGDMA-550 Da), Acrylic acid (AAc),

Potassium persulfate (KPS), Fluorescein O-methacrylate (Fluo), Polyvinyl alcohol

40-88 (PVA), Dimethyl Sulfoxide (DMSO) were all purchased from Sigma-Aldrich

(St. Gallen, CH) and used as received. The dye Methacryloxyethyl Thiocarbamoyl

rhodamine B (Rhod) was obtained from Polysciences Inc.

2.2.2 Synthesis of core-shell microgels

Core-shell microgels were prepared first by free-radical precipitation

polymerization. Reaction was carried out in a three-neck, 100 mL round-bottom

flask to which a filtered aqueous solution of 1% (w/v) total monomer concentration

and 1% (w/v) of PVA were added. This solution was heated to ~ 65 ºC while being

purged with N2 gas and stirred vigorously for ~ 1 h. Then initiator, a KPS aqueous

solution (to make a final KPS concentration of 0.06 w/v), was added and solution

turned turbid, indicating successful initiation. Rhod dissolved in dimethyl sulfoxide

(DMSO) (0.1 mL) and diluted with water (1.9 mL), was then added to the stirred

mixture at final concentration ranging from 0.1 mM (Table 2.1). The solution was

allowed to heat and stir for an additional 7 h while being purged with nitrogen gas.

To remove unreacted monomers, oligomers and surfactants microgels were dialyzed

for 15 days against distilled water, centrifuged several times, re-suspended in 25 mL

of deionized water and stored at 4 °C until further use. The rhodamine-labelled core

microgels (R1) were re-suspended in deionized water to a concentration of 10 mg/mL

and used as seeds for subsequent polymerization of additional PEGDMA cross-

linked. For the second reaction step, a solution of R1 microgels (10 mg/mL) in

deionized water (25 mL) was heated to 65 ºC under a gentle stream of N2.

Then a PEGDMA solution (0.5% w/v), previously purged with N2, was slowly

37

added. Finally, after the temperature remained stable at 65 ºC for ~ 1 h, KPS

(final concentration of 1mM) was added to initiate the polymerization (Table 2.1).

The reaction was allowed to proceed for 6 h. Microgels (R1-PEG) so obtained were

purified by analogues procedure already described and re-suspended in deionized

water. Finally, for the outer shell synthesis, a solution of R1-PEG microgels

(10mg/ml) was heated to 65 ºC, followed by the slow addition of 10 mL of aqueous

monomer solution containing PEGDMA (250 mg).

To obtain double shell microgels with different content of Fluo and AAc

(R1-FxAAcx), AAc was added in concentration ranging from 3.6-36 mM. After the

temperature remained stable at 70 ºC for ~ 1 h, 2 mL of aqueous solution of KPS

(final concentration of 1mM) was added to initiate the polymerization. Fluorescein

O-methacrylate diluted in water (2 mL) was then added to the stirred mixture at final

concentration ranging from 0.1-0.3 mM to obtain different dye amount (Table 2.1).

The reaction was allowed to proceed for 6 h. Again, also double shell microgels were

washed, re-suspended in deionized water and then stored at 4 °C prior to use until

further use.

Table 2.1: Microgel recipes. All numbers are reported as mM concentrations of the final solution.

PEGDMA RHOD FLUO AAC KPS PVA

R1 18.2 0.1 -- -- 2.2 48

R1PEG 9.1 -- -- -- 1.1 48

R1F1AAC100 9.1 -- 0.1 36 1.1 48

R1F3AAC10 9.1 -- 0.3 3.6 1.1 48

R1F3AAC100 9.1 -- 0.3 36 1.1 48

2.2.3 Microgels characterization

Particle size of microgels was evaluated by DLS measurements using Dynamic light

scattering (Malvern Zetasizer Nano ZS instrument, λ: 633 nm He-Ne laser, 173°

scattering angle).

38

The hydrodynamic diameter (Dh) was determined in the presence of 10-3 M KCl as

the background electrolyte using a dilute microgels solution 0.1% w/v. A total of

5 runs (each comprised of 3 cumulant cycles) were conducted; the experimental

uncertainties represent the standard error of the mean of 5 replicate runs.

2.2.3.1 Swelling characterization

To visualize the morphology and volumes of the microgels in the dried and swollen

state, in-situ Atomic Force Microscopy (AFM, ScanasystBruker, Germany, GmbH)

measurements were performed. In particular, for our experiments “ScanAsyst

Fluid+” (Brukerprobes) probes were chosen in order to obtain high-resolution

imaging in fluid. About thirty particles were examined for triplicate of each sample,

in both dry and wet conditions. A drop of 50 µL of microgel dispersion (⁓1 μg/mL)

was deposited on a glass wafer and dried in a vacuum oven at 40 °C, until weight

remains constant. For wet analysis, a solution of phosphate saline buffer (PBS)

10 mM at different pH was added and, for correct measurement, we waited until

microgels deposition on the surface. Images were analyzed by Gwyddion-Free SPM

(Scanning Probe Microscopy) data analysis software, applying a polynomial surface

leveling of first order and afterward particles were selected through a mask to

calculate Laplacian volume.

2.3 Results and discussion

2.3.1 Microgels characterization

Multifunctional microgels were obtained through a multistep procedure combining

free-radical precipitation polymerization and seeding polymerization, PEGDMA as

the main crosslinker. Such particles were designed to respond to the requisites of a

multiplex assay having a unique encoding system for each particle, through the

39

placement of two fluorescent dyes with emission not overlapped,

and accommodating an anchoring group on the outmost shell for subsequent

conjugations, in this case carboxyl groups (Figure 2.1).

Figure 2.1: Schematic representation of core-shell microgel synthesis obtained combining precipitation and seeded polymerizations.

Microgels obtained by each reaction step were properly characterized by DLS

analysis to determine the size distribution and evaluate the effective monodispersity.

It is known that DLS on fluorescent microparticles can be, in principle, a problem

caused by the possibility of absorption of light by the fluorophores at the wavelength

of laser. In order to minimize this undesirable effect, several dilute solutions of our

microgels were prepared and tested in order to find the best measurement conditions.

In particular, a 0.1% w/v microgels solutions were finally chosen for size

measurement. As shown in Figure 2.2, we were able to synthetize monodisperse core

microgels (R1) with a narrow size distribution (549 ± 7 nm), that can be used as seeds

for the other layers.

40

Figure 2.2: DLS analysis: particle size distribution of R1-core.

Similarly, DLS analysis of the first-shell were done, confirming monodisperse

microgels with a narrow size distribution (724 ± 16 nm) (Figure 2.3).

Figure 2.3: DLS analysis: particle size distribution of 1st shell R1PEG.

Finally, particle size distribution (Figure 2.4) and zeta potential (Figure 2.5) of a

second-shell sample (R1F1AA100) were analyzed. Final core-double shell microgels

resulted monodispersed with a narrow size distribution (976 ± 16) and Zeta potential

41

distribution of -15.4 ± 9 mV. The measurement of charge properties is clearly

important in any attempt to estimate electrostatic interactions between microgel

particles.

Figure 2.4: DLS analysis: particle size distribution of 2nd shell R1F1AA100.

Figure 2.5: DLS analysis: Zeta potential distribution of 2nd shell R1F1AA100.

42

2.3.2 AFM characterization

AFM measurements were performed in order to determine morphological

features and swelling parameters useful to probe the inside structure of microgels.

In-situ AFM measurements were conducted on all samples in dried form and at

different pH. First, we measured dried microgels, then an amount of PBS 10 mM

pH>8 was added submerging all the microgels. The system was allowed to swell

for at least 30 min and images were acquired. Afterwards, measurements in acid

conditions were performed on the same regions by exchanging the medium

(PBS 10mM at pH<3) allowing microgels to equilibrate for a period of time

(about 30 min). Polyelectrolyte hydrogels usually exhibit a variation of their

equilibrium swelling as a function of pH according to the acid–base equilibrium

for a weak acid: in acidic solution, most of the acidic units remain in the

protonated form while they are ionized under basic conditions. Indeed our

microgels respond to the different pH collapsing in acid and swelling to their

maximum size in base above their pKa (pH>8).

From a morphological point of view, R1 and R1-PEG microgels resulted fairly

spherical while all the double shell microgels assumed an irregular shape upon

the drying process. Indeed, during the drying process, microgels were dried in

vacuum at 40 °C producing an alteration of their original shape due to their

softness. Moreover, microgels in the swollen state tended to lose their spherical

structure remaining stuck on the substrate reaching on average a height far from

the original one measured by DLS in solution.

Double shell microgels resulted to be uniform in size, as reported in Figure 2.6,

where are shown example of 2D images of R1F3AAc100 in dry, swollen and

relaxed state. From those images, is possible to see more regular and spherical

shape in dry with respect to the same wet particles. The resulting profiles,

in Figure 2.6-b, revealed an irregular shape in each conditions considered

(dry and wet).

43

Figure 2.6: (a) AFM images of second shell microgels in dry, relaxed and swollen state. (b) microgels profile taken in three different direction along one microgel in dry, relaxed and swollen state. Reproduced with permission from Ref. [1], Copyright 2016 Wiley.

The measure of particle volume simply through the diameter resulted difficult and

unreliable. In the case of R1F3AAc100 microgels, the profiles taken in three different

directions along the center of a microgel were completely different in shape,

describing three different areas/volumes and bringing to a misinterpretation of the

real volume simply by the diameter. For these reasons we calculated the volume of

each particle directly form AFM images by “Laplacian volume”.

The images were leveled applying a polynomial surface leveling of first order and

creating a mask on the region of particles. In that way we were able to calculate

“Laplacian volume” that represents the volume between grain surface and the basis

surface formed by Laplacian interpolation of surrounding values. Once volumes

were calculated, swelling ratio (Q) was obtained for both swollen (Qs) and relaxed

state (Equation 2.1):

Q = Q = Equation 2.1

44

where Vd represents the dry volume, Vs the volume in the swollen state and Vr in

the relaxed state. Here we consider microgels in the swollen state when the polymer

chains are completely extended (i.e. at pH > 8 for double shell microgels and in

PBS at pH 7.4 for not responsive R1 and R1-PEG microparticles). The relaxed state

is intended the volume of microgels obtained in acid condition at pH < 3,

where the acid groups in the un-dissociated form bring to a partial collapse of the

polymer chains.

In Figure 2.7 is reported the graph of swelling ratio that shows a different trend of

Qs and Qr. As regards Qs, we can find a constant increase from R1 to R1-PEG and

the double shell microgels. Indeed the core result to have a higher density with

respect to the adlayers: indeed, in this case we reduce the concentration of

crosslinker (PEGDA) in the synthesis of the additional layers for 1% to 0.5% w/v.

On the other hand, while we were not able to calculate Qr for the not pH-responsive

microgels (R1 and R1PEG), the swelling behavior in the relaxed state of double

shell microgels showed an increasing trend that basically follows the increase of

the monofunctional monomers (Fluo and AAc). Indeed, they represent chain

extenders as the relative ratio crosslinker/monomer increase.

Furthermore considering the swelling ratio for each single spherical shell

(Qs*, Qr*) (taken as separated from the adjacent inner shell) we showed greater

differences already in the first shell, which is around 15 times higher than the one

calculated for the R1PEG. In the case of double shell microgels, the swelling

behavior of the spherical and the whole microgels followed the same trend

(Qs/r vs Q*s/r): we observed here the double contribution due to the different species

of monomers and their concentration. Indeed the R1F1AAc100 resulted more

swollen than the other double shell microgels in basic conditions wile in the relaxed

state a high degree of collapse is registered (about 4 times). In the series at higher

content of Fluo (R1F3), the swelling is decreased and the contribution of acid groups

seems negligible for both AAc100 and AAc10 samples.

45

Figure 2.7: Swelling ratio calculated in both swollen and relaxed state for microgels (Qs, Qr) and their crown (Qs*, Qr*). a: core microgel (R1); b: first shell (R1 PEG); c: second shell (R1-F1 AAc100); d: second shell (R1-F3-AAc10); e: second shell (R1-F3-AAc100). Reproduced with permission from Ref. [1], Copyright 2016 Wiley.

2.3.3 Swelling characterization

The equilibrium swelling theory allow analyzing the structure of hydrogels that do

not contain ionic moieties. As described in chapter 1, for hydrogels prepared in the

presence of water is possible to apply Peppas and Merril (Equation 1.7). For our

microgels, only in 2nd shell was possible to recognize different value of v2,s and v2,r

because of the presence of AAc that produce a pH response. For this reason, in core

and 1st shell microgels we applied the simpler Flory-Rehner equation (Equation 1.3).

Here we reported characteristic parameters used: interaction parameter PEG/water,

χ = 0.426;12 specific volume, v = 0.91 ml/g; molar volume of water, V1 = 18.1

ml/mol;12 uncrosslinked molecular weight of PEG, Mn = 550 Da. Mesh size of a gel

is strongly influenced by the degree of crosslinking and by the interaction of the

network forming polymer with the solvent. Once M̄c was calculated, the mesh size

(ξ) can be determined applying Equation 1.11. In particular, we used these values:

Cn = 4,12 the molecular weight of the repeating unit of the polymer Mr = 4412 and the

C–C bond length l = 0.146 Å.12

46

Table 2.2: Characteristic parameters of microgels: volume (dry, relaxed and swollen (μm3)), molecular weight between crosslinks calculated respectively by the Flory-Rehner (M̄c (Da))

and by Peppas-Merrill (M̄c * (Da)) formula and mesh size (ξ (nm)).

VD VR VS M̄C M̄C * ξ

R1 5.40∙10-3 -- 8.55∙10-3 46 -- 1.05

R1PEG 5.54∙10-3 -- 1.28∙10-2 115 -- 1.94

R1F1AAC100 1.57∙10-2 1.53∙10-2 5.55∙10-2 194 196 3.55

R1F3AAC10 9.45∙10-2 1.26∙10-1 2.78∙10-1 163 145 2.70

R1F3AAC100 5.98∙10-2 9.82∙10-2 1.89∙10-2 176 146 2.64

We observed that from core to first shell M̄c value increases from 46 Da to 115 Da

while ξ passes from 1 nm to 1.9 nm. For double shell microgels instead the M̄c is

194 Da, 163 Da and 176 Da while ξ is 3.5 nm, 3 nm and 3.2 nm respectively for

R1F1AAc100, R1F3AAc10 and R1F3AAc100. Then, in order to remark the AAc effect,

we applied Peppas-Merril formula to pH responsive shells (double shell microgels)

considering the relaxed state at pH < 3. In this case, we noticed that M̄c is 196 Da,

145 Da and 146 Da and ξ is 3.5 nm, 2.7 nm and 2.6 nm respectively for R1F1AAc100,

R1F3AAc10 and R1F3AAc100. Furthermore, in order to evaluate the presence of

eventual overlap region, we did a similar characterization considering only the

spherical layers (Figure 2.8).

Figure 2.8: Focus on the overlap layer between shells.

47

Results were summarized in Table 2.3. Comparing results so obtained with previous

one, we noticed a significant increase of both M̄c and ξ calculated only for the

spherical shell. Similarly appended also for R1F1AAc100 while, as regard the series

R1F3, the differences are minimal.

Table 2.3: Characteristic parameters of spherical-shell: volume (dry, relaxed and swollen (μm3)), molecular weight between crosslinks calculated respectively by the Flory-Rehner (M̄c (Da))

and by Peppas-Merrill (Mc* (Da)) formula and mesh size (ξ (nm)).

M̄C M̄C * ξ

R1 46 -- 1.05

R1PEG 274 -- 4.61

R1F1AAC100 214 252 4.57

R1F3AAC10 165 151 2.80

R1F3AAC100 181 154 2.77

Overall, the discrepancies observed in the swelling behavior could be explained

considering the presence of overlapping regions at boundaries between adjacent

layers and a certain degree of interpenetration of the shells. In order to evaluate this

overlap, we calculated volumetric fraction (χ) of single shell, considered separated

from each other in two different ways: the first one (χξ) by mesh size value

(Equation 2.2 – Equation 2.3) and the second one (χV) by volume values obtained by

AFM measurement (Equation 2.4 – Equation 2.5)

ξ / ° = ξ χ + ξ ° χ ° Equation 2.2

ξ / ° = ξ / ° χ / ° + ξ ° χ ° Equation 2.3

χ = / ° Equation 2.4

χ / ° = / °/ ° Equation 2.5

48

Table 2.4: Volumetric fraction calculated by both mesh size values (χξ ) and by volume values (χV).

χξ core χξ 1°shell χξ 2°shell χV core χV

1°shell

χV

2°shell

R1/R1PEG 0.75 0.25 -- 0.67 0.33 --

R1PEG/R1F1AAC100 -- 0.21 0.79 -- 0.23 0.77

R1PEG/R1F3AAC10 -- 0.035 0.965 -- 0.046 0.954

R1PEG/R1F3AAC100 -- 0.038 0.962 -- 0.068 0.932

When volumetric fraction calculated by mesh size showed different values from the

component obtained by volume, probably an overlap occurred and its fraction can be

estimated.

Our results showed this discrepancy suggesting the evaluation of the volumetric

fraction of overlap. Therefore, we firstly focalized on the core/1stshell layer and,

proceeding in similar way described before, we calculated the volumetric fraction of

the overlap. It resulted to be ~0.08 so we concluded that it represented a very thin

layer. Likewise, 1st/2nd shell overlap was estimated and values were approximately

the same. To sum up we can conclude that the overlap between all these three shells

exist but it is minimal.

2.4 Core-shell applications as biosensor

Core-shell microparticles so described represent an ideal support for multiplexed

bead-based assay. In particular, these microgels were used by Netti’s group13-14 as

bioassay based on optical detection (Figure 2.9).

In particular, microgels were used for direct and absolute set of miRNAs properly

chosen as cancer biomarkers. After a multistep synthesis, microgels were

functionalized with fluorescent DNA-tail, properly modified with –NH2 group that

have to react with the –COOH moiety on the microgels outer shell.

49

Figure 2.9: Graphical representation of core double shell with mechanism of miRNA detection. Adapted with permission from Ref[13]. Copyright (2015) American Chemical Society.

Moreover, our recent studies, showed the possibility to use these microparticles for

an innovative, versatile, specific and sensible assay that combine a new class of

biomarkers, the endogenous viral hCMV microRNAs, with a promising technology

based on encoded microgel beads.

Microgels so functionalized offer important advantages: first, little changes can be

followed by dynamic light scattering, which is sensitive to both swelling and surface

charge; second, microgels can be centrifuged and readily re-dispersed, which

facilitates cleaning;14-15 third, flexibility of manipulation in 3D and, finally,

microgels are generally more colloidal stable than other nanosized support particles.

This kind of microgels are continuously evolving the concepts of the liquid assays

representing a material platform with enhanced capability to overcome specific

needs.

2.5 Conclusions

In conclusion, here we report a flexible synthesis of fluorescent encoded double-shell

microgels. We successfully synthetized multifunctional microgels in a combination

50

of precipitation and seeded polymerizations demonstrating the ability of the process

to obtain well defined particles in terms of inner and outer shell chemistry.

We included carboxyl groups on the outer layer that give rise responsivity to pH

changes and the potential anchoring group for a capture molecule. The swelling

characterization, done combining AFM images and equilibrium-swelling theory,

allowed obtaining a detailed characterization of each single layer. The presence of

overlap layers was evaluated and estimated negligible. To sum up, the results

presented here, and in very recent studies of our group,7-15 suggest that such

microgels represent a valuable tool for the realization of highly hydrated

multifunctional particles with extreme flexibility and reproducibility to be used as

carrier in diagnostics.

51

2.6 References

1. Battista, E.; Mazzarotta, A.; Causa, F.; Cusano, A. M.; Netti, P. A., Core− shell microgels with controlled structural properties. Polymer International 2016, 65 (7), 747-755.

2. Birtwell, S.; Morgan, H., Microparticle encoding technologies for high-throughput multiplexed suspension assays. Integrative Biology 2009, 1 (5-6), 345-362.

3. Ulijn, R. V.; Bibi, N.; Jayawarna, V.; Thornton, P. D.; Todd, S. J.; Mart, R. J.; Smith, A. M.; Gough, J. E., Bioresponsive hydrogels. Mater Today 2007, 10 (4), 40-48.

4. Pregibon, D. C.; Toner, M.; Doyle, P. S., Multifunctional encoded particles for high-throughput biomolecule analysis. Science 2007, 315 (5817), 1393-1396.

5. Cusano, A. M.; Causa, F.; Moglie, R. D.; Falco, N.; Scognamiglio, P. L.; Aliberti, A.; Vecchione, R.; Battista, E.; Marasco, D.; Savarese, M.; Raucci, U.; Rega, N.; Netti, P. A., Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum. J R Soc Interface 2014, 11 (99).

6. Chapin, S. C.; Appleyard, D. C.; Pregibon, D. C.; Doyle, P. S., Rapid microRNA Profiling on Encoded Gel Microparticles. Angewandte Chemie-

International Edition 2011, 50 (10), 2289-2293. 7. Causa, F.; Aliberti, A.; Cusano, A. M.; Battista, E.; Netti, P. A.,

Supramolecular spectrally encoded microgels with double strand probes for absolute and direct miRNA fluorescence detection at high sensitivity. J Am

Chem Soc 2015, 137 (5), 1758-61. 8. Manikas, A. C.; Aliberti, A.; Causa, F.; Battista, E.; Netti, P. A.,

Thermoresponsive PNIPAAm hydrogel scaffolds with encapsulated AuNPs show high analyte-trapping ability and tailored plasmonic properties for high sensing efficiency. J Mater Chem B 2015, 3 (1), 53-58.

9. Jones, C. D.; Lyon, L. A., Synthesis and characterization of multiresponsive core-shell microgels. Macromolecules 2000, 33 (22), 8301-8306.

10. Langer, R.; Peppas, N. A., Advances in biomaterials, drug delivery, and bionanotechnology. AIChE Journal 2003, 49 (12), 2990-3006.

11. Peppas, N. A.; Merrill, E. W. Crosslinked poly(vinyl alcohol) hydrogels as swollen elastic networks Journal of Applied Polymer Science Volume 21, Issue 7 Journal of Applied Polymer Science [Online], 1977, p. 1763-1770. http://onlinelibrary.wiley.com/doi/10.1002/app.1977.070210704/abstract (accessed 01).

52

12. Zustiak, S. P.; Leach, J. B., Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules 2010, 11 (5), 1348-1357.


of the American Chemical Society 2015, 137 (5), 1758-1761. 14. Aliberti, A.; Cusano, A. M.; Battista, E.; Causa, F.; Netti, P. A., High sensitive

and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles. Analyst 2016, 141 (4), 1250-1256.

15. Causa, F.; Aliberti, A.; Cusano, A. M.; Battista, E.; Netti, P. A. In Microgels

for multiplex and direct fluorescence detection, 2015; pp 952919-952919-7.

53

Chapter 3

PEGDA hydrogels

as potential engineered-based assay

Abstract. In recent years, development of new detection system, in several fields, has rapidly increased and so there is a growing interest in the research of novel customizable materials. The possibility to control the network structure as well as the swelling behavior is important in the synthesis of materials with tunable final properties. The aim of this chapter is to define a set-up to attain a tailored hydrogel-based material that combine both molecular filter and capture element capability. PEGDA-bulk hydrogels were synthetized by UV radical photopolymerization using different polymer concentrations (10-15-20% w/v). In order to test the molecular filter capability, diffusion studies of several probes (sulforhodamine G and dextrans) with different hydrodynamic radii, were carried out using NMR technique. This analysis showed that only 10%-15% polymer allow obtaining appropriate network for our purposes. Moreover, fluorimetric analysis were fulfilled using functionalized PEGDA-hydrogels, properly obtained polymerizing PEGDA with a methacrylate oligonucleotide. These studies were carried out to evaluate the oligonucleotide diffusion, probe density, efficiency and kinetic of hybridization. Results confirmed that only 10-15% allow probe diffusion. Furthermore, we demonstrated that high probe density obstructs the hybridization. Therefore, we developed a set-up for functionalized hydrogels that merge the best condition of probe density and diffusion transport to allow oligonucleotide capture.

This chapter is part of an article in preparation: A. Mazzarotta, T.M. Caputo, L. Raiola, E. Battista, F. Causa, P.A. Netti.- “PEGDA hydrogels as

potential engineered-based assay.”

54

3.1 Introduction

In order to accomplish an engineered device useful for molecular recognition is

important to find a suitable material and investigate the critical parameters that

affect the efficiency of recognition. Because of the concentration of molecules

represent an obstacle for recognition, the possibility to use hydrogels as

molecular filter is important in order to reduce crowding effects within the

hydrogels. Therefore the modulation of both the network structure and capture

element concentration allow to obtain a tunable material for efficient molecule

capture.

Hydrogels, due to their potentiality such as biocompatibility and high water

content, have been of great interest as biomaterials for many years.1-3

The network is crosslinked by chemical bond or physical entanglements, which

give them the ability to swell or shrink, altering the overall structure.4-8

The most common characterization of this material is based on three parameters:

the polymer volume fraction in the swollen state, v2,s, the molecular weight

between crosslinks, M̄c, and the mesh size ξ. The study of these parameters and

the suitable modulation to obtain an engineered material, results to be

fundamental to understand the network structure that allow us to predict the final

properties of our materials.

In this chapter, we focus on the chemical and physical properties of hydrogels

that primarily affect the final properties of materials. Particularly we evaluate the

parameters that play an important role in the probe diffusion inside the network.

The performances of a hydrogel are dependent to a large extent on its bulk

structure and on the interaction with solvent molecules.9 The knowledge and the

control at molecular level of hydrogel structure is then a crucial issue in the

network customization to permit the diffusion of selected target, based on the size

and the chemical content. In fact, hydrogels can work as molecular filter

representing a physical barrier for large molecules and repelling unspecific

55

binding (antifouling property) (Figure 3.1). By the contrast, a specific binding

can be achieved adding, during polymerization or in a post modification step,

a molecular catcher inside the bulk or on the surface.

Figure 3.1: Schematic representation of engineered bulk hydrogels.

Several methods for evaluating bulk hydrogel properties are well studied in the

last years.4-7, 9-11 In particular, the swelling ratio and polymer volume fraction in

the swollen state can be measured through equilibrium swelling experiment.7

Then the molecular weight and the mesh size can then be obtained by a theoretical

model for swelling of polymer hydrogels.12

The structure of hydrogels can be analyzed by the Flory-Rehner theory,

subsequently modified by Peppas and Merrill, which developed a theoretical

framework that has significant success in describing the hydrogel swelling

behaviour.4, 11

In addition to these parameters, for many applications, diffusion into the network,

controlled by the mesh size of the gel, is important to be investigated.

Several information, in fact, can be gained by studying the diffusion of probe

molecules through hydrogel networks.13-14 In the absence of any specific

interactions between the probe and the network, the diffusivity of the probe

reflects the network structure.14-16

Common techniques used to study probe diffusion include source–sink

techniques,17 fluorescence recovery after photobleaching (FRAP),14, 18 and pulsed

56

field gradient-NMR (PFG-NMR).13, 16, 19-21 The key for PFG-NMR analysis is the

fact that NMR data render information on the chemical nature as well as on the

molecular mobility of an observed component. Nuclear magnetic resonance

spectroscopy, most commonly known as NMR spectroscopy, is a technique that

exploits the magnetic properties of certain atomic nuclei. It determines the

physical and chemical properties of atoms or the molecules in which they are

contained. It relies on the phenomenon of nuclear magnetic resonance and can

provide detailed information about the structure, dynamics, reaction state, and

chemical environment of molecules.

The advantage of this analysis is that works in the absence of concentration

gradients, studying instead the chaotic motion of molecules driven by

intermolecular collisions.13 Diffusion times over a large range, usually from few

milliseconds up to seconds, are available via PFG-NMR techniques and can be

chosen arbitrarily by selecting the correct attenuation parameters.13

The study of the probe diffusion on these short timescales, allow obtaining much

information on the microscopic structure of materials.13, 22-26 Furthermore,

this technique has the potential to characterize water binding and mobility in a

directly quantifiable manner, and has been commonly used in studies of the

water–polymer interaction. In particular, it is possible to measure the water self-

diffusion coefficient (D) that is directly related to the potential for water to leave

a hydrogel.27

The identification and study of critical parameters that affect the efficiency of

recognition result to be fundamental for the application of these hydrogels for

assay. For this application, in fact, not only the network structure affect the

adequacy of the assay but also the concentration of molecules, representing an

obstacle for recognition. Appropriate diffusion studies are fundamental to

evaluate the molecules crowding effect and avoid the unwanted obstruction.

57


3.2.1 Materials

Poly(ethylene glycol)diacrylate (PEGDA-700 Da), Deuterium oxide (D2O),

Sulforhodamine G, Albumin-fluorescein isothiocyanate conjugate (BSA), Ethanol

and Diethyl ether were purchased from Sigma-Aldrich (St. Gallen, CH) and used as

received. Crosslinking reagent 2-Hydroxy-2-methylpropiophenone (DAROCUR

1173) was provided from Ciba. Texas Red labelled dextrans with different molecular

weight (3 kDa, 10 kDa and 40 kDa) were supplied by Thermo Fisher Scientific. DNA

oligonucleotides were purchased from Metabion with HPLC purification. NMR

tubes, properly designed for gel samples, were supplied by New Era Enterprises, Inc

and OptiPlate-96 F HB were purchased by PerkinElmer.

3.2.2 Synthesis of bulk-hydrogel

The bulk-hydrogels were synthesized using a UV free radical

photopolymerization. The process involves three basic steps (Figure 3.2). First, a

free radical must be formed through the initiation step. For the PEGDA system

used in this work, the initiator is darocur, which forms a free radical under UV

light (Figure 3.2 a). The next step is known as propagation, where the free radical

from the initiator comes into contact with the end of a PEGDA molecule and

reacts with the carbon-carbon double bond in the acrylate functional group

(Figure 3.2 b). This step produces a second free radical species, which can go on

to react with more PEGDA polymers propagating the crosslink. The final step in

the process of this polymerization is termination, which occurs when two radical

species meet and a bond forms between them. A general reaction scheme for a

difunctional polymer forming a network is reported in (Figure 3.2 c).

58

Figure 3.2: UV free radical photopolymerization: (a) Initiation mechanism,

(b) Propagation mechanism, (c) Formation of a crosslinked polymer network.

Our mixture was composed of poly(ethylene glycol) diacrylate (PEGDA, 700 Da),

at different concentrations in Milli-Q water and darocur used as initiator. Each

hydrogel sample was prepared dissolving photoinitiator in 10 mL of polymer

solution, obtaining a darocur final concentration of ~0.1% v/v (Table 3.1).

The reaction mixture was strongly mixed and then purged with high purity nitrogen

for 5 minutes to remove the excess of oxygen that may interfere with the free radical

polymerization. The reaction mixture was then carried out in 10 mL test tubes and

illuminated with UV lamp (λ = 365 nm and power-lamp = 10 W) for 5 minutes

leading to complete polymerization. For NMR studies, we reduced the total reaction

volume to 1 mL of deuterated water (Table 3.1). Polymerization was performed

directly in special NMR tubes designed for gel samples. Hydrogel samples were than

expelled from the NMR tube and put in a solution of D2O for the next 24h to remove

all the unreacted materials. Hydrogels were dried in oven at 30°C for few hours,

swelled with 1 mL of probe solution and finally put in the NMR tube for the

59

measurement. In particular, different probes, sulforhodamine G and several dextrans

(3 kDa, 10 kDa and 40 kDa) were dissolved in D2O to reach a final concentration of

1 mg/mL.

Moreover, functionalized bulk were synthesized using UV free radical

photopolymerization between PEGDA and oligonucleotide, properly modified with

methacrylamide moieties. Propagation step of the reaction between polymer and

methacrylate oligonucleotide is highlighted in Figure 3.3.

Figure 3.3: UV free radical photopolymerization between PEGDA

and methacrylate oligonucleotide.

Several recipes with different PEGDA and oligonucleotide concentrations were

prepared and tested. The mixture was composed of PEGDA (MW 700 Da)

at different concentrations (10-15-20% w/v) in buffer solution, darocur as initiator

and fluorescent oligonucleotide methacrylate (F-DNA-Tail) at different

concentrations (1μM-5μM) (Table 3.1). Our buffer solution was obtained adding

1 x PBS and NaCl 200 mM in milliQ water. The reaction mixture was strongly mixed

and then purged with high purity nitrogen for 5 minutes to remove the excess

of oxygen that may interfere with the free radical polymerization. Then, 100μL

of the mixture was carried out in 96-optiplate, illuminated with the same UV lamp

used before for 5 minutes leading to complete polymerization.

60

Table 3.1: Bulk hydrogels recipes.

PEGDA

(% w/v) SOLVENT

DAROCUR

(% v/v) OLIGONUCLEOTIDE

B-10% 10% H2O 0.1% -- B-15% 15% H2O 0.1% -- B-20% 20% H2O 0.1% -- B-25% 25% H2O 0.1% -- B-30% 30% H2O 0.1% --

N-10% 10% D2O 0.1% -- N-15% 15% D2O 0.1% -- N-20% 20% D2O 0.1% --

F1-10% 10% Buffer

solution 0.1% 1 μM

F1-15% 15% Buffer

solution 0.1% 1 μM

F1-20% 20% Buffer

solution 0.1% 1 μM

F2-10% 10% Buffer

solution 0.1% 5 μM

F2-15% 15% Buffer

solution 0.1% 5 μM

F2-20% 20% Buffer

solution 0.1% 5 μM

3.2.3 Bulk-hydrogel characterizations

Several characterization, attained due to NMR and fluorometer measurements,

were accomplished on different bulk samples to define the structural and diffusion

properties of these materials in both PEG-bulk and functionalized-hydrogels.


Swelling characterization of hydrogels were carried out in order to calculate polymer

volume fraction in swollen state (v2,s). It was evaluated starting from the mass of

hydrogel measured in both swollen and dry conditions. Once polymerized, hydrogels

were removed from the glass tubes, uniformly cut into samples of short cylindrical

61

shape and then immersed in an excess of milli-Q water in order to remove the

unreacted reagents. Water was changed several times for the next 24 h and samples

were then swollen overnight in water at room temperature. Then cylindrical parts

were gently wiped with a filter paper and weighted to obtain the mass of swollen

hydrogel (ms). For dried measurement, in order to obtain a total dehydration,

hydrogels were properly grinded and lyophilized overnight, so the mass of dry

samples were recovered (md). Therefore, we were able to determined hydrogel

volume in both swollen and dry state, using Equation 3.1:28

v , = Equation 3.1

were ρpolymer = 1.12 g/ml and ρpolymer = 1 g/ml. Moreover, the water contents of the

gels, which represent the mass fraction of water (Fwater) in the swollen condition,

were calculated using Equation 3.2:

F = Equation 3.2

where mswollen and mdry are, respectively, the weight of the swollen and dry hydrogels.

Then, using equations described in chapter 1, polymer volume fraction

(Equation 1.6), molecular weight between crosslinks (Equation 1.3) and mesh size

(Equation 1.11) were calculated.

3.2.3.2 NMR measurement

NMR analysis was performed using an Agilent 600 MHz (14 Tesla) spectrometer

equipped with a DD2 console. 1H-1D-NMR spectra were recorded at 300 K with

64 scans to obtain a good signal to noise ratio. Diffusion spectra were recorded at

300 K using a Dbppste (DOSY bipolar pulse pair stimulated echo) pulse sequence

Figure 3.4.

62

Figure 3.4: DOSY sequence29.

An array of 15 pulse field gradient values from 0:98 to 63:7 Gauss/cm were used

with a gradient pulse length (δ) of 2 ms. Diffusion delay (Δ) in ms was optimized for

each sample to obtain a ratio of 1:0.2 between I(Gmin) and I(Gmax), where I(Gmin)

is the intensity of the NMR peak at the minimum gradient value and I(Gmax) is the

intensity of the NMR peak at the maximum gradient value. Diffusion coefficients

were calculated using the formula reported in Equation 3.3:

, = − − Equation 3.3

where I and I0 are the spin echo amplitudes in the presence and in the absence of a

magnetic field gradient pulse, respectively; γ is the gyromagnetic ratio of the given

nucleus, Hz/T, and g is the magnetic gradient, T/m. NMR data were processed and

analyzed using VNMRJ4 software.

3.2.3.3 Fluorescent measurement

In order to evaluate the best concentration of both polymer and oligonucleotide and

to check the effective hybridization of Fluorescent-DNA-Tail and BHQ-strand,

fluorescence analysis were done. Fluorescence spectra of fixed amount of labelled

oligonucleotides were collected in a 1 cm path length cuvette with a Horiba

JobinYvon model FluoroMax-4 fluorometer equipped with a Peltier temperature

63

controller. The conjugated and unconjugated sequences were excited at 647 nm with

a slit width of 5 nm, and emission spectra were collected from 667 to 750 nm with a

slit width of 5 nm. Then, using a 2300 EnSpire multilabel reader (Perkin-Elmer,

Waltham, MA) several tests on the functionalized hydrogel samples were done.

Fluorescence was measured from the top of the plate, with excitation wavelength of

647 nm, and emission wavelength at 667nm, height 9mm and 500 number of flash.


3.3.1 Swelling characterization

The bulk-hydrogels were synthesized using a UV free radical photopolymerization.

Several recipes, using different polymer concentration (10-15-20-25-30 % w/v),

were tasted in order to obtain various network structure with different molecular

filter capability. In Table 3.2 are summarized water content (Fwater) and all

fundamental swelling parameters (v2,s, Q, M̄c, ξ) for each preparation. Firstly, we

noticed a range of water content from 91% up to 73% with increasing of polymer

concentration. Moreover, changing the PEGDA concentration, polymer volume

fraction in the range of 0.08-0.24 was obtained. As expected, Q, M̄c and ξ decreased

with increase of polymer concentration. In fact, the equilibrium-swelling ratio

decreased from 12.6 to 4.1 as the crosslink density increased. The molecular weight

between crosslinks decreased from 340 to 259 Da as the percent polymer fraction

increased from 10 to 30% (w/v). Mesh size were achieved in the range of

2.6-1.6 nm, as reported in Figure 3.5. All these results are attributable to the fact

that as the amount of polymer present for crosslinking in the hydrogel increases,

the degree of crosslinking increases and thereby decrease both the average

molecular weight between crosslinks, the equilibrium-swelling ratio and mesh size.

64

Table 3.2: Properties of bulk hydrogels of PEGDA. All values were calculated from averaged measured values.

PEGDA

10%

PEGDA

15%

PEGDA

20%

PEGDA

25%

PEGDA

30%

Fwater 91.2% ± 0.1% 87.2% ± 0.1% 82.7% ± 0.1% 78.2% ± 0.1% 73.5% ± 0.1%

v2,s (∙10-3) 79.2 ± 0.9 116.2 ± 1.8 157.5 ± 0.5 199.3 ± 0. 5 243.4 ± 0.4

Q 12.63 ± 0.14 8.61 ± 0.14 6.349 ± 0.018 5.017 ± 0.012 4.109 ± 0.008

M̄c (Da) 340.43 ± 0.21 329.35 ± 0.66 311.62 ± 0.23 288.24 ± 0.30 259.06 ± 0.31

ξ (nm) 2.652 ± 0.011 2.299 ± 0.014 2.022 ± 0.003 1.800 ± 0.002 1.597 ± 0.002

Figure 3.5: Mesh size values for different bulk-PEGDA concentrations.

These results confirm that the polymer concentration permit to optimize the network

density for the specific purpose.

3.3.2 NMR: diffusion studies

3.3.2.1 Hydrogels polymerization

One-dimensional 1H-NMR spectra of PEGDA/darocur solution were recorded

before and after UV treatment to confirm that polymerization was completed.

In Figure 3.6 1H-NMR spectra of PEGDA/darocur solution before polymerization is

65

shown. Very intense peaks at 3.73 (f), 3.85 (e) and 4.38 (d) ppm are typical of PEG

main chain methylene group while peaks a, b and c in the olefinic region

(6.0 to 6.6 ppm) refer to PEGDA terminals acrylic protons. Peaks in the aromatic

region (a’, b’ and c’ from 7.5 to 8.2 ppm) and singlet at 1.45 ppm (d’) become,

respectively, from aromatic and methyl groups protons of darocur. Broad peak at

4.65 ppm is the H2O residual signal. Peak integrations demonstrate chemical

structures of solution components and their related amount in the mixture. Moreover,

signals attribution was confirmed by 1H-NMR spectra of individual components,

reported in Appendix A.

Figure 3.6: 1H-NMR spectrum of PEGDA/darocur solution before polymerization with signal attribution and related peak integration.

In Figure 3.7 is shown H1-NMR spectra of the same PEGDA/darocur solution after

polymerization. Complete polymerization was confirmed by the disappearing of the

PEGDA acrylic signals. In addition, the very broad peak from 4.5 to 3.5 ppm showed

the transition of PEG chains from a very flexible situation (solution) to a less flexible

66

state (hydrogel). Relative decrease of the darocur peak intensities also confirmed that

a large amount of the initiator was consumed in the polymerization reaction. Again,

the broad peak at 4.65 ppm is the H2O residual signal.

Figure 3.7: 1H-NMR spectrum of PEGDA/darocur solution after polymerization.

3.3.2.2 Water diffusion

DOSY experiments were used to study the diffusion of molecules, with different

size, in hydrogels samples prepared with a PEGDA concentration of 10%, 15% and

20% (w/v). Firstly, we evaluated the water self-diffusion coefficient. Measurement

resulted to be 24.55 ± 0.11 (∙10-10 m2/s), in agreement with literature data.30

Furthermore, diffusion coefficient of solvent in the first hydration shell of hydrogels

sample was calculated. Results indicated that water diffusion coefficient was

affected by the PEGDA concentrations as shown in Table 3.3 and Figure 3.8.

(Fitting curve reported in Appendix B).

67

Table 3.3: Diffusion coefficients (10-10 m2/s) of water in PEGDA hydrogels. PEGDA 10% PEGDA 15% PEGDA 20%

Water 18.84 ± 0.03 16.05 ± 0.02 15.08 ± 0.13

Figure 3.8: 2D DOSY of water (4.645 ppm) in hydrogels bulk with different PEGDA concentrations (20% in blue, 15% in red and 10% in black).

Finally, we evaluated the relation between diffusion coefficients and water content

previously calculated. As reported in Figure 3.9, diffusion coefficients increased

with increasing of Fwater. In particular, as the number of water molecules per unit mass

of polymer increases, the ability of water molecules to diffuse increase,

approximately linearly, toward that of free water (100% Fwater). These results are

comparable with data obtained by McConville et al.27 In their work, was reported a

measurements of the water self-diffusion coefficient for a set of nine commercially

available contact lens hydrogels, both at equilibrium water content (EWC) and as a

function of reduced water content, using the pulsed field gradient NMR method.

68

Figure 3.9: Diffusion coefficient versus water content for hydrogels sample.

3.3.2.3 Probes diffusion

Probes were properly chosen to simulate the behavior and diffusion of biological

molecules. In particular, the smallest dextran (3 kDa) has hydrodynamic radius

comparable with oligonucleotide (that represent molecule that we want to capture).

On the other hand, 40 kDa dextran and blood proteins, such as albumin, have similar

hydrodynamic radii and represent molecules that we want to exclude from our

network. In order to validate our choice, hydrodynamic radii of sulforhodamine G

and dextrans (3 kDa, 10 kDa, 40 kDa) were calculated using Einstein-Stokes

equation (Equation 3.4), where KB is the Boltzmann’s constant, T is the absolute

temperature and η is the viscosity of D2O and D0 is their diffusion coefficient in

water, measured by DOSY-NMR.

= Equation 3.4

Results (Table 3.4) showed that probes diffusion coefficients in D2O (D0) were in

the range of 0.5-3.7 [10-10 m2/s] and their hydrodynamic radii had values between

3.9 and 0.6 nm. These results were comparable with the range size between small

oligonucleotides and large molecules that we need to investigate for biological

69

studies. Our results are comparable with hydrodynamic radii and diffusion

coefficients reported in literature, obtained both by NMR31 and other different

techniques such as diffusion measurement through microporous membrane filters,32

fluorescence correlation spectroscopy (FCS)33 and fluorescence recovery after

photobleaching (FRAP).34

Table 3.4: Diffusion coefficient of different probes in D2O and respective hydrodynamic radii.

D0 [10-10 m2/s] R0 [nm]

Sulforhodamine G 3.67 ± 0.06 0.61 ± 0.01

Dextran 3kDa 1.60 ± 0.03 1.34 ± 0.01

Dextran 10kDa 0.91 ± 0.01 2.41 ± 0.01

Dextran 40kDa 0.557 ± 0.008 3.92 ± 0.03

In Figure 3.10 were reported, in double logarithmic plot, diffusion coefficients of

these probes as function of their molecular weight. Our trend (black point) show a

linearity in agreement with literature data of Callaghan et al (red triangle) which

studied a self-diffusion of several dextrans both in water and in a polydisperse

polymer system.31 These results showed the effective relation between molecular

weight and diffusion coefficient which strictly decrease with increasing of probe

molecular weight.

Figure 3.10: Probe diffusion coefficient as function of its molecular weight. Comparison between data obtained by this thesis (•) and literature data (▲).31

70

Afterwards, probes diffusion coefficients in bulk hydrogels (10%, 15%, 20% w/v)

were calculated, as reported in Table 3.5.

Diffusion coefficient of small molecules, as sulforhodamine G (SR-G), should not

be affected by a very large network structure and their values should be comparable

with free diffusion of probe in water.35 On the other hand, for bigger molecules as

dextrans with different molecular weight, we expect a decreasing trend of diffusion

coefficient values from the water solution to the highest polymer percentage

hydrogel. Results, reported in Figure 3.11, confirmed those hypotheses.

Table 3.5: Diffusion coefficient (∙10-10 m2/s) of water and different probes in both water and PEGDA hydrogels.

WATER PEGDA 10% PEGDA 15% PEGDA 20%

Water 24.55 ± 0.11 18.84 ± 0.03 16.05 ± 0.02 15.08 ± 0.13

Sulforhodamine G 3.67 ± 0.06 3.14 ± 0.15 3.11 ± 009 3.10 ± 0.24

Dextran 3kDa 1.60 ± 0.03 0.87 ± 0.12 0.63 ± 0.02 0.46 ± 0.04

Dextran 10kDa 0.91 ± 0.01 0.27 ± 0.02 0.234 ± 0.001 0.120 ± 0.005

Dextran 40kDa 0.557 ± 0.008 0.160 ± 0.001 0.140 ± 0.006 0.090 ± 0.003

Figure 3.11: Diffusion coefficient of several probes in both water and hydrogels with different PEGDA concentrations.

71

Moreover, the effect of the polymer concentration on the reduction of diffusion

coefficients was highlighted in Figure 3.12, where D/D0 was plotted against polymer

concentration.

D/D0 values of water and sulforhodamine G, the smallest probe, resulted to be

constant (⁓85%) for all PEGDA concentrations; by the contrast, dextrans probe

showed a significant mobility reduction, related with the polymer concentrations.

In particular, for 3 kDa dextran, D/D0 decreased from 55 % in the wider network up

to 30% in the tighter one. Instead, both 10 kDa and 40 kDa had similar trend, between

30% and 15%, that confirms the mobility of these bigger molecules resulted to be

deeply affected by the polymer network.

Figure 3.12: Effect of the polymer concentration on the reduction of diffusion coefficients (D/D0).

Furthermore, in Figure 3.13 we plotted diffusion coefficients of dextrans probes as

function of their hydrodynamic radius, in both water (D0) and in three different

polymer network (DPEGDA10%, DPEGDA15%, DPEGDA20%). For water solution and for

10-15% (w/v) of polymer concentrations, diffusion coefficients linearly decreased

with hydrodynamic radii. Instead, probes diffusion coefficients in PEGDA 20%

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(w/v) changed its trend showing that, in this network, diffusion of dextrans 10 kDa

and 40 kDa was strictly hindered.

Figure 3.13: Diffusion coefficients of dextrans probes as function of their hydrodynamic radius obtained in water (•), PEGDA 10% (•), PEGDA 15% (•) and PEGDA 20% (•).

Pluen et al.34 described in their work the diffusion of several macromolecules in

agarose gels, applying different models to characterize the network.

With our studies, instead, we demonstrated the possibility to simplify their

characterization combining DOSY diffusion measurement with swelling

characterization, obtaining several information about our polymer network.

To sum up, NMR analysis show that hydrogels whit different PEGDA concentrations

are able to exclude big molecules by their low diffusion coefficient.

Moreover, we can modulate the diffusion of the probe modifying the mesh size of

the network, changing the PEGDA concentrations. In particular, dextran 3 kDa

seems to be still able to pass through all the gels we made while dextrans 10 kDa and

40 kDa showed a very low diffusion also in the wide network.

As a matter of fact, our results indicate that it is possible to make a tunable

PEGDA hydrogel with specific control of its network and use it as molecular filter.

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3.3.3 Preliminary study for the assay

Preliminary studies for the detection assay optimization were fulfilled using a probe

scheme already tested in previous work of our group.36 The probe scheme was

schematically reported in Figure 3.14

Figure 3.14: Scheme of oligonucleotide detection.

where fluorescent T-DNA is a short fluorescent DNA tail (12 nt), labelled at 5’ end

with ATTO 647N and modified at 3’ with methacrylamide spacer to allow its

covalent bound with polymer network. A Quencher strand (21 nt), internally

modified with a Black Hole Quencher (BHQ), was used as probe diffusion.

When the tail hybridize, the BHQ comes in close proximity with the fluorophore and

fluorescence quenching occurs.

For our studies, we used three DNA-sequences as reported in Table 3.6:

fluorescent T-DNA that was covalently bounded with polymer network;

complementary sequence (C) for the hybridization studies and non-complementary

sequence (N) as control.

Table 3.6: Sequence and thermodynamic parameters of the DNA probes used in this study.

probe sequence (5'-3') length (nt)

Fluorescent T-DNA (F-DNA-Tail) TG AAA TCG GTT A 12

Complementary sequence (C) T AAC CGA TTT CG ATG GTG CTA 21

Non-complementary sequence (N) GAG CUA CAG UGC UUC AUC UCA 21

74

In order to check the quenching efficiency of fluorescent tail with quencher strand,

we firstly studied this system in 1M PBS solution. We tested different tail

concentrations (5 μM - 0.1 μM) and BHQ-strand was added 1:1 respect to the tail.

Results showed a quenching percentage from ~93% to 88% (Figure 3.15).

Figure 3.15: Quenching percentage in solution for different oligonucleotide concentrations.

Then, we studied the ATTO 647 response to UV treatment, measuring fluorescence

pre and post polymerization, with and without photo-initiator. Results, schematized

in Figure 3.16, showed that, in the presence of the darocur, fluorescence intensity

significantly decrease post UV-treatment (~20% lower).

Figure 3.16: ATTO 647 response to UV treatment, with and without darocur in solution.

75

Then we tested the probe solution, 5 μM, within gels. Here, we reported the

fluorescence intensity for the upper and lower polymer concentration (20% and 10%

(w/v)). Firstly, we verified that, also in this case, UV-treatment produce a decrease

of fluorescence intensity. Furthermore, we added BHQ strand and repeated the

analysis. Comparing values of quenching percentage for the two different polymer

concentrations, we attained a similar quenching percentage values (~75%).

However, washing the bulk with PBS solution and re-analyzing the samples, we

found that in PEGDA 20% bulk, the quenching was not effective (Figure 3.17-a).

In this case, in fact, the BHQ was near enough to collide with fluorophore with a

consequent fluorescence decrement (dynamic quenching) but not enough to create a

stable duplex and, therefore, after washing the fluorescence was recovered.

By the contrast, in PEGDA 10%, the network was sufficient wide to allow the

formation of stable complex between oligonucleotide strands, so that the tail and

quencher came in proximity and quenching occurred, even after several washes

(Figure 3.17-b).

Figure 3.17: Fluorescence intensity of ATTO-BHQ in PEGDA 20% (a) and 10% (b).

In order to confirm that the narrow mesh size allowed only a dynamic quenching

without the hybridization of duplex, we tasted a bulk with 50% PEGDA using both

complementary (C) and non-complementary (N) BHQ-probes.

As reported in Figure 3.18, after the adding of C-BHQ we obtained a ~70%

quenching that strongly decreased at ~3% after washing. Moreover, also adding an

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N-BHQ we reached a high quenching percentage, proportional with the N-BHQ

concentrations. Therefore, we can conclude that both narrow mesh size and high

BHQ-strand concentrations do not allow an effective duplex hybridization.

Figure 3.18: Quenching percentage with both complementary (C-BHQ) in green and several not complementary concentrations (N-BHQ) in grey.

Finally we studied the kinetic of quenching for three different PEGDA

concentrations (10%-15%-20% w/v) analyzing the effect of both F-DNA-Tail and

BHQ-strand concentrations.

In particular, all tests were carried out with 1 μM and 5 μM F-DNA-Tail with

BHQ-strand 1x and 5x with respect of Tail. The three PEGDA samples showed

different trend, confirming results previous described caused by the crowding of both

polymer and oligonucleotides.

As regards PEGDA 10%, Figure 3.19 showed that quenching percentage increased

with increasing of both F-DNA-Tail and BHQ concentrations. In particular, using

F-DNA-Tail 1μM with BHQ in 1x (a), we reached an 80% of quenching after 120h.

As expected, this quenching percentage was lower than that obtained in solution.

Furthermore, adding BHQ 5x (b) we reached earlier, after 72h, the same 80 %.

Finally, increasing both F-DNA-Tail and BHQ, we obtained 80% quenching in less

than 48h (d).

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Figure 3.19: Quenching kinetic in PEGDA 10% with several oligonucleotides concentrations: (a) F-DNA-Tail 1μM- BHQ 1x; (b) F-DNA-Tail 1μM- BHQ 5x; (c) F-DNA-Tail 5μM- BHQ 1x; (b) F-DNA-Tail 5μM- BHQ 5x.

On the other hand for PEGDA 15%, only with F-DNA-Tail 5mM and BHQ 1x was

possible to reach, after 96h, an 80% quenching (b).

In fact, with constant F-DNA-Tail, a lower BHQ concentration was not enough to

provide a good quenching while an increase of F-DNA-Tail produced a crowding

effect that obstacle the effective hybridization.

78


Finally, for PEGDA 20% we reached very low values of quenching for all sample

preparations. Moreover, the decrease of quenching percentage post washing

confirmed the not efficient hybridization.

79


These preliminary studies gave us several information: firstly, polymer

concentrations higher than 15% PEGDA were not suitable for probe diffusion that

resulted to be hindered. Similarly, in order to reduce the crowding effect and permit

the hybridization, we needed to use 1 μM Tail.

Finally, the significant fluorescence reduction caused by UV-treatment suggested

changing the probe scheme (see chapter 4) using a not-fluorescent tail.

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3.4 Conclusions

In this chapter, we focused on the synthesis and characterization of PEGDA-based

bulk hydrogels. Swelling characterization showed the possibility of modulating mesh

size and swelling parameters, changing the polymer concentration, in order to

achieve different filter ability.

NMR analysis confirmed the opportunity to use hydrogels as molecular filter.

In particular, 10% and 15% polymer concentrations resulted to be suitable for our

purposes. In fact, these networks allowed the diffusion of molecules with size

comparable with our probe (oligonucleotides) and excluded big molecules with size

similar with proteins and antibody.

Moreover, fluorimetric analysis gave us important information regarding the ratio

between polymer and oligonucleotides concentrations to let the efficient

hybridization. In particular, 10% and 15% PEGDA concentrations showed a suitable

network for both oligonucleotide diffusion and effective hybridization. In addition,

results showed that for 10% of polymer we can obtain a good quenching percentage

also for the highest oligo concentration tested. On the other hands, for 15% of

polymer, we cannot use oligonucleotide concentration higher than 1 μM, because of

crowding effect.

To sum up, our results showed the possibility to obtain functionalized and engineered

materials, with a tunable network, that allow to combine both molecular filter and

capture element capabilities. In this way, we can improve these materials to simplify

the detection analysis of several target. These reported studies inspire us to scale-

down this knowledge into micrometric-scale, in order to reduce sample volume and

associated costs, as reported in the following chapter.

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3.5 Appendix A

Here are reported and compared 1H-NMR spectra of individual components darocur (IV) and PEGDA (III) and mixture pre (II) and post- polymerization (I).

82

3.6 Appendix B

Interpolation curves fitting NMR- DOSY results fitted by Equation 3.3 for water

diffusion in PEGDA 10-15-20% (w/v), respectively.

84

3.7 References

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biomolecules to radiation-grafted hydrogels on inert polymer surfaces. ASAIO

Journal 1972, 18 (1), 10-16. 2. Hoffman, A. S., Hydrogels for biomedical applications. Advanced drug

delivery reviews 2012, 64, 18-23. 3. Zalipsky, S.; Harris, J. M., Introduction to chemistry and biological

applications of poly (ethylene glycol). ACS Publications: 1997. 4. Peppas, N. A.; Merrill, E. W., Crosslinked poly (vinyl alcohol) hydrogels as

swollen elastic networks. Journal of Applied Polymer Science 1977, 21 (7), 1763-1770.

5. Peppas, N.; Huang, Y.; Torres-Lugo, M.; Ward, J.; Zhang, J., Physicochemical foundations and structural design of hydrogels in medicine and biology. Annual review of biomedical engineering 2000, 2 (1), 9-29.

6. Peppas, N. A.; Keys, K. B.; Torres-Lugo, M.; Lowman, A. M., Poly (ethylene glycol)-containing hydrogels in drug delivery. Journal of controlled release

1999, 62 (1), 81-87. 7. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R., Hydrogels in

biology and medicine: from molecular principles to bionanotechnology. Advanced materials 2006, 18 (11), 1345-1360.

8. Kopecek, J., Hydrogels: From soft contact lenses and implants to self‐assembled nanomaterials. Journal of Polymer Science Part A: Polymer

Chemistry 2009, 47 (22), 5929-5946. 9. Peppas, N.; Bures, P.; Leobandung, W.; Ichikawa, H., Hydrogels in

pharmaceutical formulations. European journal of pharmaceutics and

biopharmaceutics 2000, 50 (1), 27-46. 10. Peppas, N. A.; Khare, A. R., Preparation, structure and diffusional behavior of

hydrogels in controlled release. Advanced drug delivery reviews 1993, 11 (1-2), 1-35.

11. Bryant, S. J.; Anseth, K. S., Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly (ethylene glycol) hydrogels. Journal

of Biomedical Materials Research Part A 2002, 59 (1), 63-72. 12. Flory, P. J., Principles of polymer chemistry. Cornell University Press: 1953. 13. Wallace, M.; Adams, D. J.; Iggo, J. A., Analysis of the mesh size in a

supramolecular hydrogel by PFG-NMR spectroscopy. Soft Matter 2013, 9 (22), 5483-5491.

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14. Sun, J.; Lyles, B. F.; Yu, K. H.; Weddell, J.; Pople, J.; Hetzer, M.; Kee, D. D.; Russo, P. S., Diffusion of dextran probes in a self-assembled fibrous gel composed of two-dimensional arborols. The Journal of Physical Chemistry B

2008, 112 (1), 29-35. 15. Branco, M. C.; Pochan, D. J.; Wagner, N. J.; Schneider, J. P., Macromolecular

diffusion and release from self-assembled β-hairpin peptide hydrogels. Biomaterials 2009, 30 (7), 1339-1347.

16. Cohen, Y.; Avram, L.; Frish, L., Diffusion NMR spectroscopy in supramolecular and combinatorial chemistry: an old parameter—new insights. Angewandte Chemie International Edition 2005, 44 (4), 520-554.

17. Cao, S.; Fu, X.; Wang, N.; Wang, H.; Yang, Y., Release behavior of salicylic acid in supramolecular hydrogels formed by l-phenylalanine derivatives as hydrogelator. International journal of pharmaceutics 2008, 357 (1), 95-99.

18. Scalettar, B. A.; Hearst, J. E.; Klein, M. P., FRAP and FCS studies of self-diffusion and mutual diffusion in entangled DNA solutions. Macromolecules

1989, 22 (12), 4550-4559. 19. Colsenet, R.; Söderman, O.; Mariette, F., Pulsed field gradient NMR study of

poly (ethylene glycol) diffusion in whey protein solutions and gels. Macromolecules 2006, 39 (3), 1053-1059.

20. Gao, P.; Fagerness, P. E., Diffusion in HPMC gels. I. Determination of drug and water diffusivity by pulsed-field-gradient spin-echo NMR. Pharmaceutical research 1995, 12 (7), 955-964.

21. Kwak, S.; Lafleur, M., Self-diffusion of macromolecules and macroassemblies in curdlan gels as examined by PFG-SE NMR technique. Colloids and

Surfaces A: Physicochemical and Engineering Aspects 2003, 221 (1), 231-242. 22. Gagnon, M.-A.; Lafleur, M., Comparison between nuclear magnetic resonance

profiling and the source/sink approach for characterizing drug diffusion in hydrogel matrices. Pharmaceutical development and technology 2011, 16 (6), 651-656.

23. Gagnon, M.-A.; Lafleur, M., Self-diffusion and mutual diffusion of small molecules in high-set curdlan hydrogels studied by 31P NMR. The Journal of

Physical Chemistry B 2009, 113 (27), 9084-9091. 24. Escuder, B.; LLusar, M.; Miravet, J. F., Insight on the NMR study of

supramolecular gels and its application to monitor molecular recognition on self-assembled fibers. The Journal of organic chemistry 2006, 71 (20), 7747-7752.

25. Shapiro, Y. E., Structure and dynamics of hydrogels and organogels: An NMR spectroscopy approach. Progress in Polymer Science 2011, 36 (9), 1184-1253.

26. Kärger, J., NMR self-diffusion studies in heterogeneous systems. Advances in

Colloid and Interface Science 1985, 23, 129-148.

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27. McConville, P.; Pope, J., A comparison of water binding and mobility in contact lens hydrogels from NMR measurements of the water self-diffusion coefficient. Polymer 2000, 41 (26), 9081-9088.

28. Litzenberger, A. L., A microfluidic method to measure diffusion in hydrogels. 2010.

29. Johnson Jr, C. S., Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Progress in Nuclear Magnetic Resonance

Spectroscopy 1999, 34 (3-4), 203-256. 30. Holz, M.; Heil, S. R.; Sacco, A., Temperature-dependent self-diffusion

coefficients of water and six selected molecular liquids for calibration in accurate 1H NMR PFG measurements. Physical Chemistry Chemical Physics

2000, 2 (20), 4740-4742. 31. Callaghan, P. T.; Pinder, D., A pulsed field gradient NMR study of self-

diffusion in a polydisperse polymer system: dextran in water. Macromolecules

1983, 16 (6), 968-973. 32. Lebrun, L.; Junter, G.-A., Diffusion of dextran through microporous

membrane filters. Journal of membrane science 1994, 88 (2-3), 253-261. 33. Weiss, M.; Elsner, M.; Kartberg, F.; Nilsson, T., Anomalous subdiffusion is a

measure for cytoplasmic crowding in living cells. Biophysical journal 2004, 87 (5), 3518-3524.

34. Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A., Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations. Biophysical

journal 1999, 77 (1), 542-552. 35. Cheng, Y.; Prud'Homme, R. K.; Thomas, J. L., Diffusion of mesoscopic probes

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of the American Chemical Society 2015, 137 (5), 1758-1761.

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Chapter 4

Engineered PEGDA-microparticles by

microfluidics: oligonucleotide functionalization

for selective miRNA 143-3p detection in serum

Abstract. Droplet microfluidics is the most powerful technology used to produce and manipulate monodisperse droplets. This technique addresses the need for lower costs, shorter times, and higher sensitivities. In such a way, we were able to synthesize high controlled microparticles, directly functionalized in flow, eliminating time-consuming labelling steps and their associated costs. Therefore, here we present a versatile and innovative assay that combine biomarker detection with engineered and functionalized microparticles obtained by microfluidic. In particular, firstly we report microfluidic optimization to achieve a high monodispersity and spherical shape. Then, both swelling characterization and diffusion studies, by CLSM, were carried out to reach a specific control of both network structure and probes diffusion. In particular, our studies were fulfilled using poly(ethyleneglycol) microparticles functionalized with DNA-Tail, properly designed for the direct detection of miRNA 143-3p (associated with several cardiovascular disease). The detection system is based on double-stranded displacement assay and do not require any sample manipulations. Therefore, we demonstrated the possibility to attain a high control of synthesized microparticles and the possibility to modulate both recipe and microfluidic parameters to obtain specific molecular filter ability and target recognition. This Chapter is part of an article in preparation: A. Mazzarotta, T.M. Caputo, E. Battista, F. Causa, P.A. Netti.- “Engineered PEGDA-microparticles by

microfluidics: oligonucleotide functionalization for selective miRNA 143-3p

detection in serum.”

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4.1 Introduction

In recent years, application of biosensor has rapidly increased and so there is a

growing interest in the research of innovative tunable materials. Several studies of

hydrogels-based technologies has been widely fulfilled with important improvement

in their synthesis strategies. These materials are used for many different applications,

in particular in biotechnology field for diagnostic,1-4 drug delivery,5-8 and tissue

engineering.9-12 In fact, due to the possibility of tune their size, shape and chemistry

and for their biocompatibility, they represent ideal candidates for biosensing

applications.

As demonstrated in chapter 3, it is possible to synthesize engineered hydrogels,

with tunable network, to achieve a detection system for specific target.

In this chapter, we scaled-down this knowledge into the micro-scale, producing high

controlled microparticles for detection of biomolecules.

4.1.1 Microfluidic technique

Hydrogel microparticles for sensing purposes should be synthesized with high

control in size, shape and chemical composition, trying to match the requirements of

liquid bioassays as regard the encoding, the capturing system and mass transport.

Conventional techniques for the syntheses of hydrogel microparticles are

represented by dispersion, precipitation and emulsion polymerization

procedures.13-15 These methods have intrinsic limits, in fact the shape of such

particles is restricted to spheres and often are not enough monodisperse. Moreover,

biomolecules are incompatible toward organic solvents and high temperature

conditions needed. Recently, with the improvement of microfabrication methods,

hydrogel microparticles can be obtained using microfluidic device, achieving a tuned

size and shape with a good chemical-physical properties control. In particular,

droplet microfluidics facilitate large production of microparticles with a rate greater

than 105 particles per hours and a narrow size monodispersity less than 5–6 %, not

89

possible with conventional methods, even though limited by the need of chemical

mixtures of immiscible fluids and surface-compatible with microfluidic devices.16

This approach represents the most promising methods for the production and the

functionalization of monodisperse particles at low cost and reagent consumption,

with higher reproducibility and saving time.17

Microfluidic techniques, based on droplet generation methods, have been wildly

studied since 2005 as continuous on-chip production of water-in-oil emulsions based

on the break-off of droplets in two-phases through different geometry: T-junction,

flow focusing and co-flowing (Figure 4.1).18-20

Figure 4.1: Droplet generation in three microfluidic devices: T-junction, flow focusing and co-flowing geometry.

Several variables such as flow rates, dimensions of geometry, viscosity of the fluid,

surface tension and capillary number strongly affect the droplet formation.

The control of these parameters is fundamental to generate monodisperse and stable

emulsion. In particular, the capillary number (Ca), that represents the relative effect

of viscous forces versus surface tension acting across the interface between

two immiscible fluids, is an adimensional number that can allow predicting the

emulsion stability.

Many studies has been carried out to define critical values of Ca. Choi et al.21 found

a limited range of Ca (0.5 x 10-2 –0.5 x 10-1) and a flow rate of disperse phase

(0.2 - 1.1 μL/min) where stable and monodisperse emulsions can be generated.

Celetti et al.22 performed simulation in a flow-focusing droplet generator showing

90

that each of the parameters that influence Ca, affect the size of particles and the flow

configurations after the junction. Nunes et al.23 analyzed in detail the transition

between dripping and jetting regimes (Figure 4.2). In their works,24-29 was

summarized that stable and monodisperse emulsions can be produced in T-junction

device in Ca range of 10-3 – 10-1 and a flow rate ratio 10-2 – 100.

In the jetting regime, the droplets are generated not only due to the natural growth of

an interfacial instability but also because of the viscous forces applied by the

continuous phase fluid. In the dripping regime, instead, the combination of capillary

instability and viscous drag is proposed to argue the mechanism of the generation of

drops; in this case the size of the droplets is much smaller than the dimensions of the

channel of the continuous phase fluid.23 Surely, the dimension of geometry influence

Ca optimal range.

Figure 4.2: Schematic of flow regimes in T-junction microfluidic devices

in both dripping (a) and jetting (b) regime.

Microparticles can be functionalized directly in flow or in a second step, employing

strategies in droplet microfluidics. In fact, they can be engineered with several

biological entities such as protein, peptide or nucleic acids5 for the detection of

biomarkers in complex fluids.1 The possibility to functionalize hydrogels directly

during synthesis step let us to find new strategies to eliminate time-consuming

labelling steps and their associated costs (Figure 4.3).

91

Figure 4.3: Engineered microparticles for specific target detection.

4.1.2 Biomarkers detection

Biomarkers cover a broad range of biochemical entities, such as proteins, nucleic acids,

small metabolites, sugars and tumor cells found in the body fluid. They are widely used

for risk assessment, diagnosis, prognosis, and for the prediction of treatment efficacy and

toxicity.30-31 In biomarkers context, miRNAs have attracted a growing attention as

confirmed by the increasing of publication regarding micro RNA during the past

20 years (Figure 4.4). Due to their immense regulatory power, the variation of

their levels can be utilized as potential biomarkers for the diagnosis and prognosis

of a variety of diseases.32-35

Figure 4.4: Histogram showing the increase in publications related to the keyword “microRNA” during the past 20 years. PubMed data.

92

In particular, several studies identified miRNA-143-3p as important transcriptional

and post-transcriptional inhibitors of gene expression. The integrity of transcriptional

and post-transcriptional regulatory circuit is essential for the maintenance of cardiac

homeostasis. Nowadays, cardiovascular disease is the predominant cause of human

morbidity and mortality in developed countries. miRNA-143-3p contribute to many

cardiovascular processes such as embryonic stem cell differentiation, cardiomyocyte

proliferation, endothelial responses to shear stress36-44 and play an essential role in

the function and formation of the cardiac chamber. Moreover, dysregulation of

miRNA-143-3p physiological level is associated with many cardiovascular disease

including heart failure, myocardial ischemia, congenital heart disease,

atherosclerosis and hypertension.45

Conventional techniques for the detection of oligonucleotides, such as microarray

analysis46-47, polymerase chain reaction (PCR, qRT-PCR)48 show several drawbacks

due to preliminary steps needed (extraction and amplification) resulting in time-

consuming analysis49. Due to high sequence homology, complex secondary

structures, and low concentration levels, an accurate and robust quantification of

circulating miRNA biomarkers in blood is still a major challenge for early stage,

metastatic or recurrent diseases.

In this chapter, we propose an innovative bead-based assay, highly specific, which

combine miRNA-143-3p detection with engineered and functionalized

microparticles. Overcoming conventional techniques, our detection system is based

on double-stranded displacement assay and do not require any sample manipulations.

As concerns the assay, several experimental conditions can affect the final assay

times.50 Several studies have been conducted to optimize DNA hybridization. Ravan

et al.51 demonstrated that efficiency of hybridization could be influenced by many

constraints such as electrostatic repulsion between oligonucleotide strands and the

steric hindrance between neighboring DNA probes. All these interactions, in fact,

reduce both the efficiency and the kinetic of DNA hybridization.

Stevens et al.52 focused on the determination of time required for particles

93

hybridization to reach equilibrium. They underlined that DNA hybridization on

microparticles is temperature-dependent, with reactions coming to equilibrium faster

at high temperatures than at low temperatures. In particular, in their work, was shown

that, when all other parameters are equal, time required for reaction to reach

equilibrium goes from 1 h at 50 °C to 24 h at 20 °C. Finally, Wong’s group50

examined both the ionic strength and probe sequences effects on the hybridization

time. In their work, were reported the kinetics of the assay at different salt

concentrations. Time required generally decreases with the salt concentration.

In particular their results showed a rapid decrease in the range of 5-50 mM NaCl and

a slowly decrease for NaCl concentrations over 100 mM. Moreover, also the length

of two sequences that hybridize play an important role in the kinetics of the assay.

It was evaluated the half-time of assay for different probe length. This time goes

from 4 minutes for 10-base probe up to 3 h for the 23-base.

To sum up, nucleic acid hybridization kinetic is affected by surface probe density,

surface charge, conformation and sequence of probe strands and by the curvature of

the solid surface. Moreover, incubation temperature and ionic strength also sway the

hybridization time. Therefore, several parameters have to be considered to evaluate

the time required to reach hybridization equilibrium.


4.2.1 Materials

Poly(ethylene glycol) diacrylate (PEGDA-700 Da), Light mineral oil (LMO),

Sorbitan monooleate (SPAN 80), Polyethylene glycol sorbitan monolaurate

(TWEEN 20), Ethanol and Diethyl ether, Albumin-fluorescein isothiocyanate

conjugate (BSA), Anti-Human IgG (whole molecule)-FITC antibody produced in

goat (IgG) were purchased from Sigma-Aldrich (St. Gallen, CH) and used as

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received. Crosslinking reagent 2-Hydroxy-2-methylpropiophenone (DAROCUR

1173) was supplied from Ciba. Phosphate Buffered Saline Tablets were purchased

from MP Biomedicals. Texas Red labelled dextrans with different molecular weight

(3 kDa, 10 kDa and 40 kDa) were supplied by Thermo Fisher Scientific. DNA and

RNA oligonucleotides were provided from Metabion with HPLC purification.

Human serum was supplied by Lonza. μ-Slide were provided by Ibidi. Tube and

special DNA-LoBind tubes were provided by Eppendorf.

4.2.2 Synthesis of functionalized microparticles

Microparticles were synthesized using a microfluidic device bought from Dolomite.

The chip is a glass microfluidic device with hydrophobic coating. It was designed

for generating droplets, both with T-junction and Flow focusing geometry.

For our purpose, we used the first geometry that consists of two inputs for the

continuous and disperse phase, a narrow orifice where the two opposite channels

converge, and an output (Figure 4.5).

The dimensions of the device are 22.5 mm × 15.0 mm × 4 mm (length × width ×

thickness) with wide channels cross-section of 100μm × 300 μm (depth × width) and

channels cross-section at junction of 100 μm × 105 μm (depth × width) (Figure 4.6).

Figure 4.5: The Droplet Junction Chip and its Chip Interface H for fluidic connection.

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Figure 4.6: Dolomite chip: geometry and size.

Microparticles were synthesized using light mineral oil (LMO) containing non-ionic

surfactant Span 80 (5 % v/v) as a continuous phase and a water solution of

poly(ethylene glycol) diacrylate (PEGDA, MW 700 Da) (10-15-20 % w/v) with

photoinitiator darocur (0.1-1% v/v with respect to the total volume) as dispersed

phase. In order to functionalize microparticles, methacrylate oligonucleotide was

added in the dispersed phase to reach final concentration of 1μM into the water

solution. Similar UV free radical polymerization already described for bulk

(chapter 3), occurred. In Figure 4.7. was reported our experimental set-up.

Droplet emulsions were obtained adding in the first channel water solution and

continuous phase in the other. Solutions were injected using high-precision syringe

pumps (neMesys-low pressure) to ensure stable flow and reproducibility.

This system was mounted on an inverted microscope (IX 71 Olympus). The droplets

formation was monitored using an objective with 5x magnification and

0.12 numerical aperture and recorded with a CCD camera ImperxIGV-B0620M

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that allow to record up to 259 frame per second. Once emulsion was formed,

it was crosslinked outside the chip using an UV lamp at 365 nm wavelength at

different power lamp (42-2000 mW) for ⁓ 2 minutes obtaining a complete

polymerization. Thereafter, microparticles were collected in an eppendorf and

exceeded oil phase was removed. Then particles were washed several times with

different solvents (diethyl ether, ethanol and milliQ water-tween solution

(0.05 % v/v)) to remove the residual oil. After washing, microgels were stored at

room temperature in buffer solutions until further use. Our buffer was obtained

mixing 1x PBS, NaCl 200 mM and tween-20 at 0.05% v/v in milliQ water.

Figure 4.7: Set-up for functionalized microparticles generation.

4.2.3 Microparticles characterization


Approximately 100 microparticles diluted in 20 μL were loaded onto μ-slide

18 well-flat. Images in both swollen and dry conditions were collected with

CLSM microscope (CLSM Leica SP5- with an Objective HC PL FLUOROTAR

20x0.5 DRY and a scan speed of 8000 Hz). Then, images were analyzed by ImageJ

software to obtain diameter and calculate volume of microparticles in both

conditions. Therefore, using equations described in chapter 1, polymer volume

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fraction (Equation 1.6), molecular weight between crosslinks (Equation 1.3) and

mesh size (Equation 1.11) were calculated.

4.2.3.2 Morphological characterization

Morphological characterization was done collecting images by SEM (scanning

electron microscope) and CLSM (confocal laser scanning microscope). SEM

measurements were performed on a FE-SEM Ultra Plus (Zeiss) microscope at 20

kV. For sample preparation, the microparticles solution was fixed on a microscope

slide, air-dried and then sputtered with a 10 nm thin gold layer.

4.2.3.3 Diffusion characterization

Analysis were carried out following fluorescence intensity changes by CLSM.

10 μL of microparticles solution, containing approximately 100 microparticles,

were loaded onto μ-slide 18 well-flat with 10 μL of probe solution (dextrans 3 kDa,

10 kDa, 40 kDa, Target143, BSA and IgG with final concentration 2 μM).

Samples were illuminated at CLSM Leica SP5 using appropriate wavelength

(λex 543-488 nm) and fluorescence images of microparticles were collected.

4.2.3.4 Target detection

Fluorescence analysis, performed by CLSM with λex 633 nm, were also used for the

target detection. All experimental steps were performed at room temperature in

buffer phosphate solution (1x PBS, NaCl 200 mM and tween-20 at 0.05% v/v).

Non-fluorescent particles were analyzed adding in μ-Slide VI 0.4 a solution

constituted by diluting 1 μL of DNA-Tail particles solution in 30 μL of buffer.

As regard Tail hybridization step, 100 μL of microparticles-Tail solution

(~25∙103 particles and ~6 pmol of Tail), were loaded in DNA-low binding tube with

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F-DNA 10x (~60 pmol). Solution was kept under stirring until use for three days and

then washed several times with our buffer.

Finally, for the CLSM experiment, 1 μL of this solution was diluted in 30 μL

of buffer, added in μ-Slide VI 0.4 and therefore the fluorescence intensity

was evaluated.

Concerning displacement step, several samples containing 2μL of microparticles-

Tail-F solution (~330 particles and ~60 fmol of Tail), were loaded in DNA-low

binding tubes with different Target concentrations (from 100 μM to 50 pM in buffer

solution). Analogously to the previous step, solution was kept under stirring and

washed several times with buffer. For the fluorescent analysis, 1 μL of this solution

was diluted in 30 μL of buffer. To test the system in complex fluid, 2μL of

microparticles-Tail-F solution were put in contact with Target-Human serum

solution. The latest was prepared adding 200 μL of serum and 300μL of our buffer

with Target 2 uM.


4.3.1 Probe design

Probe used in this chapter was designed for selective and specific miRNA

fluorescence detection according to a double strand (ds) displacement assay. In order

to overcome problems connected with fluorescence reduction caused by

UV-treatment, observed and described in chapter 3, we modified our scheme as

reported in Figure 4.8. In particular, based on the miRNA 143-3p sequence (21 nt),

we designed the ds probes formed by a tail (T-DNA, 12 nt), modified

with methacrylamide spacer at the 3’end, for covalent bound with polymer network

and a longer fluorescent DNA sequence (F-DNA, 21 nt), labelled with ATTO 647N

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at 5’ end. F-DNA sequence is partially complementary to T-DNA and totally

complementary to the specific target.

The probe design implies that, when the F-DNA and T-DNA partially hybridize,

the fluorescence occurs. In presence of the target, the F-DNA and the target hybridize

so that low fluorescence background signal is registered.

Figure 4.8: New scheme showing the mechanism of miRNA detection based on ds displacement assay.

Thermodynamic parameters such as melting temperature, stability at physiological

ionic strength and folded fraction at assay condition were evaluated using

appropriate tools (IDT-Integrated, D. N. A. "Technologies. OligoAnalyzer 3.1.

Web." (2014)).

The length of the tail was optimized to obtain an appropriate difference in free

energy. Thermodynamic parameters of the probes used for this work were reported

in Table 4.1. TFhyb represents the free energy gained from the partially

complementary T-DNA/F-DNA duplex; FTargethyb is the free energy gained from

the fully complementary Target/F-DNA duplex; Δdisplacement is the free energy

gained after T-DNA/F-DNA de-hybridization and Target/F-DNA hybridization.

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Table 4.1: Sequence, length and thermodynamic parameters of the DNA probes and RNA targets.

miRNA 143-3p probe

probe sequence (5'-3') length

(nt) ΔG (Kcal/mol)

T-DNA GCA CTG TAG CTC 12 0

F-DNA TGA GAT GAA GCA CTG TAG CTC 21 TFhyb = -12.76

Target GAG CUA CAG UGC UUC AUC UCA 21 FTargethyb = -22.62

Δdisplacement = 9.86

4.3.2 Microparticles: optimization of microfluidic parameters

Several physical parameters such as geometry, flow rates, viscosity of the fluid,

capillary number (Ca) and surface tension influence the droplet generation.14

Hence, the control of these parameters is important for the production of

monodisperse and stable emulsion. In particular, we focused on the effect of capillary

number (Ca) on the emulsion stability. Ca represents the relative effect between the

viscous stress and surface tension acting across an interface between two immiscible

fluids.18

Firstly, we investigated the influence of Ca, modulated by varying flow rate of the

continuous phase (Qoil), on emulsion stability. As reported in Figure 4.9, fixed our

geometry device, for Qoil values higher than 15 μL/min unstable emulsion were

generated and increasing more this value, the flow of polymer solution reversed into

channels. When the value of Ca was in a limited range 0.05 ∼ 0.20 and the value of

Qoil between 6 and 10 μL/min, the polymer phase broke into a stable emulsions.

Precise control of particle size and monodispersity are fundamental for many

applications of microparticles.Microfluidic device allows this control over a wide

range of sizes. For this reason, we evaluated the influence of Qoil on the droplet size.

Different microgel sizes were reported in Figure 4.9 with a range of sizes from 20 to

100 μm. Droplets diameter were calculated in flow during the droplet production

before and after polymerization showing similar results.

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Figure 4.9: Diagram as a function of continuous phase flow rate (Qoil) and capillary number (Ca).

Two different conditions are reported: stable (green) and unstable (red) emulsion.

Moreover, dripping and jetting regimes were investigated (Figure 4.10) changing

flow rate ratio (Qoil/QPEGDA), as reported in Figure 4.11.

Our results showed three different flow conditions after the junction: stable dripping,

stable jetting and unstable emulsions. It was difficult to generate stable emulsion in

dripping mode at high flow rates of polymer solution. In fact, W/O emulsion could

not separate uniformly from the junction due to the unstable hydrodynamic pressure

of PEGDA phase.

Figure 4.11 also show different emulsion size obtained for different flow rate ratio.

Figure 4.10: Droplet generation in dripping (a) and jetting (b) regimes.

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Figure 4.11: Diagram as a function of flow rate ratio (QOIL/QPEGDA) and dispersed phase flow rate

(QPEGDA) operating in jetting (yellow rectangle), dripping (blue rectangle) and unstable regime (red rectangle).

In order to stabilize droplets against uncontrolled coalescence, the use of a surfactant

was required. These molecules are often added on purpose in order to facilitate

the creation and transport of drops and to prevent droplet coalescence.

In the emulsification process, the value of interfacial tension displays a strong

dependence on the local surface coverage with surfactant molecules and play an

important role on droplet size and stability53.

4.3.3 Microparticles: morphological characterization

Microparticles were constantly monitored during production step, images were

recorded and their size was measured just after emulsion formation using Droplet

103

monitor software. It allowed obtaining several information about the droplet

generation. In particular data analysis show the average droplet size and their

distribution, the spacing between droplet and droplet rate.

For the final particles synthesis, specific flow rates were chosen: 0.5 μL/min for

dispersed phase and 6.5 μL/min for continuous phase. In such a way, we were able

to achieve a high throughput production of particles with a droplet rate of

⁓40 particles/sec.

Moreover, once polymerized, particles were collected, washed and characterized by

optical and electronic microscope. Images so obtained (Figure 4.12a-c) confirmed

that our microparticles were monodisperse and spherical, with a diameter of

79.2 ± 1.17 μm (Figure 4.12b).

Figure 4.12: Optical image (a) of monodisperse microparticle

and relative particle size distribution (b); (c) SEM image of microparticles.

4.3.4 Microparticles: swelling characterization

Firstly, in order to optimize experimental set-up and recipe, we evaluated the effect

of lamp power and photo initiator concentration on the mesh size. For these studies,

microparticles without oligonucleotide were prepared. Figure 4.13 show the mesh

size values of PEGDA 15% microparticles, with three different darocur final

concentrations (1-0.5-0.1% v/v), polymerized with several lamp power (from 42 to

2000 mW).

104

Figure 4.13: Effect of both lamp power and darocur concentration

on mesh size of microparticles with PEGDA 15%.

Results showed that for lower darocur concentration (0.1%), particles

polymerization occurred only with lamp power values higher than 850 mW.

Increasing the photoinitiator concentration up to 1%, particles can be well

polymerized also with lamp power of 42 mW.

All particles were characterized and mesh size values resulted to be comparable for

all power lamp values and for all darocur concentrations. Therefore, both variables

did not affect the final mesh size of microparticles. For subsequent characterizations

and for microparticles production, we decided to use the lower darocur final

concentration (0.1%) with power lamp of 850 mW.

As regards a complete swelling characterization, several samples obtained using

polymer concentrations of 10-15-20 % w/v, were prepared in order to characterize

different network structure and to compare these results with bulk one presented in

chapter 3. In particular, swelling parameters (v2,s, Q, M̄c , ξ) were calculated.

(Table 4.1). As expected, same trend previously described was obtained also for

microparticles. In particular, changing the PEGDA concentration, polymer volume

fraction in the range of 0.008-0.206 was obtained. The equilibrium-swelling ratio

decreased from 11.42 to 4.87 with PEGDA increasing. The molecular weight

between crosslinks decreased from 338 to 283 Da as the crosslink density increased.

105

With these recipes, mesh size in the range of 2.55-1.77 nm was achieved

(Figure 4.14), resulting between 4-11 % lower with respect to mesh calculated for

bulk. This difference can be expected because of the difference in both synthesis

procedure and in mesh size analysis.

Table 4.2: Properties of PEGDA microparticles. All values were calculated from averaged measured values.

PEGDA 10% PEGDA 15% PEGDA 20%

V2,s 0.088 ± 0.007 0.125 ± 0.007 0.206 ± 0.014

Q 11.42 ± 0.87 7.99 ± 0.46 4.87 ± 0.33

Mc (Da) 338 ± 2 325 ± 4 283 ± 12 ξ (nm) 2.55 ± 0.10 2.23 ± 0.07 1.77 ± 0.09

Figure 4.14: Mesh size values for different microparticles-PEGDA concentrations.

Then we added methacrylate DNA-Tail in the mixture reaction. In Table 4.3 were

reported mesh size values (ξ) of 20% PEGDA with different oligonucleotide

concentrations. Results showed that there was not significant effects.

Table 4.3: Mesh size of PEGDA (15%w/v) microparticles with different T-DNA concentrations.

No Oligo T-DNA 400 nM T-DNA 1 μM T-DNA 10 μM

ξ (nm) 2.23 ± 0.07 2.12 ± 0.05 2.18 ± 0.14 2.10 ± 0.07

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For subsequent analysis, functionalized microparticles were synthesized using only

15% polymer concentration. In fact, results showed in chapter 3 demonstrated that

higher PEGDA concentration were not suitable for an efficient hybridization,

because of the narrow network. On the other hand, microparticles synthesized with

10% polymer resulted to be too soft and not enough resistant to washing procedure.

Therefore, several recipes using 15% polymer and different methacrylate DNA-Tail

concentrations (400nM- 1μM - 10μM) were prepared to evaluate the effect of

oligonucleotide concentration on the network structure.

To sum up, this swelling characterization demonstrate that is possible to obtain

different molecular filter capability regulating the polymer concentration.

For the specific purpose of selected miRNA, we decided to synthesized particles with

15% polymer concentration and T-DNA 1μM, which combine both network

structure and stability required for the detection. As reported in chapter 3,

higher T-DNA concentrations produced a crowding effect that obstacle the effective

hybridization.

4.3.5 Diffusion studies: CLSM analysis

Diffusion studies were carried out using 15% PEGDA microparticles with and

without 1μM DNA-Tail.

Diffusion percentage was evaluated following the fluorescence intensity changes by

CLSM images. In particular, several probes were tested: dextrans 3 kDa, 10 kDa,

40 kDa, Target143, BSA and IgG all, of them with final concentration of 2μM.

In correspondence of this concentration value, detected solute intensities inside the

hydrogel and in the surrounding solution were proportional to dye concentration.

Intensities values were reported in graph as ratio between the fluorescence intensity

inside the microparticles (IIN) and fluorescence in the surrounding gel (IOUT).

107

As reported in Figure 4.15, we noticed that ⁓85% of both 3 kDa and 10 kDa dextrans

were able to spread into microparticles. A smaller percentage, ⁓40%, of 40 kDa and

oligonucleotide are capable to go into the hydrogel.

Finally, only a 10% of proteins (BSA and IgG) were detected into the network.

These results were reported for functionalized microparticles; the same results were

obtained also for microparticles not functionalized, with not significant differences

in fluorescence values.

Figure 4.15: Time lapse of fluorescence intensity (IIN/IOUT) of several probes. All values reported show a standard deviation of 10%.

Then fractal dimension (df) was evaluated for dextrans probes used for this work.

This parameter gave us information about their rigidity, in particular for a rigid

sphere, df=3.0, for a linear Gaussian polymer coil (random walk) df=2.0, and df < 2

for polymers with excluded-volume interactions.54 Fractal dimension was defined by

the scaling law (Equation 4.1)55:

M ∝ r Equation 4.1

where M represents their molecular weight and r their size, expressed as

hydrodynamic radius. Figure 4.16 showed a double logarithmic plot of the probe

molecular weight versus their hydrodynamic radius.

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Figure 4.16: Dextrans molecular weight as function of its hydrodynamic radii.

The fractal dimension calculated for our dextrans was df ~2.4 and it was in agreement

with previously published results.54, 56 The df > 2 is thought to arise from partial

branching of dextrans. Our results suggested that dextran probes were not rigid

particles and therefore they can spread into a narrow network, as occurred in our

microparticles.

Moreover, we calculated partition coefficient (k) as the ratio between the

concentration of the solute in the external aqueous solution equilibrated with the gel

(COUT) and the concentration of solute in the gel (CIN) per unit volume of gel.57

In correspondence of our probes concentrations, detected solute intensities inside the

hydrogel and in the surrounding solution were proportional to dye concentration.

Consequently, the partition coefficient was given by the ratio of solute intensity in

the microparticles to that in the loading solution. In Figure 4.17 are reported partition

coefficient of dextrans as function of their hydrodynamic radii.

109

Figure 4.17: Dextrans partition coefficient as function of hydrodynamic radius.

Plotted values showed that larger solutes exhibit progressively smaller partition

coefficients, indicative of size exclusion from the water domain of our

microparticles.

Finally, we reported I/IMAX versus time in order to compare diffusion behavior of all

probes inside the microparticles. Experimental points were fitted with Equation 4.2,

and reported in Figure 4.18.

Figure 4.18: Time lapse of fluorescence intensity (I/Imax) of several probes. All values reported show a standard deviation of 10%.

110

= 1 − Equation 4.2

where τ is the extrapolated value that represent time required to achieve 63% of

maximum intensity. In such a way, we were able to determine τ for each curve,

as reported in Table 4.4.

Table 4.4: Extrapolated values of τ for each probes.

Probes τ (min)

Dextran 3 kDa 2.1 ± 0.1


Oligonucleotide 5.7 ± 0.3


BSA 28.4 ± 1.2

IgG 40.4 ± 2.0

Results showed that smaller probes such as dextrans 3 kDa and 10 kDa required

about 2-3 minutes to accomplish 63% of maximum value. Oligonucleotide needed

⁓6 minutes while dextrans 40 kDa required twice the time of the latter. Finally, both

proteins needed longer times, approximately 30-40 minutes.

Because of τ is inversely proportional to the diffusion coefficient, these data

confirmed the molecular size exclusion of our polymer network.

To sum up, our results showed that it is possible to use our engineered microparticles

as molecular filter that allow the target to go in and exclude the majority of big

molecules as proteins.

111

4.3.6 miRNA 143-3p detection

As reported in paragraph 4.3.1, our double strand probe consisted of a short

methacrylate DNA-Tail (T-DNA, 12 nt), covalently bound to the polymer networks,

and a longer fluorescent DNA sequence (F-DNA, 21 nt). The latter was partially

complementary to DNA-Tail and totally complementary to a specific target. Target

identification occurred by double strand displacement assay (Figure 4.19).

Figure 4.19: Mechanism of Target detection.

In particular, results reported below were referred to large excess of Target

concentration (2 μM). 1μl of T-DNA particles solution was loaded into μ-Slide,

analyzing by CLSM their fluorescence intensity.

As showed in Figure 4.20a-Figure 4.21a, only a low background fluorescent signal

is registered. Then 100 μL of T-DNA particles solution were used for tail

hybridization. The longer F-DNA (10x with respect to the tail concentration) was

added toT-DNA particles solution. This was stirred until used and washed with

buffer solution before next measurement.

Hybridization efficiency was monitored for three days until the equilibrium was

reached. Then 1μL of the washed solution was diluted in 30 μL of buffer solution

and loaded in μ-Slide for fluorescence intensity analysis.

112

Images showed a significant increase in microgels fluorescence and, therefore,

hybridization between DNA-Tail and F-DNA has occurred (Figure 4.20b and

Figure 4.21b).

The required time needed to reach equilibrium is not unusual considering our

experimental conditions. In fact, even if Stevens et al.52 demonstrated that was

possible to reduce time required for hybridization increasing both temperature and

tail concentration, we suffer of some limitations. Indeed, due to the low melting

temperature of our hetero-duplex (Tm= 45°C), we can not raise the temperature to

enhance hybridization efficiency. Moreover, regarding the oligonucleotide

concentration, as showed in chapter 3, we can not use more than 1 μM of

methacrylate tail for PEGDA 15% (w/v) because of the crowding effect observed.

Finally, another parameter that can be modified to enhance the hybridization time,

is the ionic strength50. However, ore buffer was already optimized, with the addiction

of 200 mM of NaCl, to reach the best assay conditions.

In order to evaluate the displacement, 2 μL of the Tail/F solution were added to the

solution that was constantly stirred until equilibrium was reached. Then washing

steps were done to remove F-DNA/target complex.

Analogously to the previous procedure, 1μL of the washed solution was diluted in

30 μL of buffer solution and loaded in μ-Slide. Figure 4.20c-Figure 4.21c showed

that, in the presence of target, fluorescence strongly decrease confirming that

displacement occurred.

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Figure 4.20: CLSM images of the three fundamental steps of target identification:

(a) T-DNA, (b) adding F-DNA, (c) displacement in presence of the Target.

Figure 4.21: Fluorescence intensity of (a) T-DNA, (b) adding F-DNA,

(c) displacement in presence of the Target in buffer solution.

Moreover, we tested several target concentrations from 100 μM to 50 pM. Results,

reported in Figure 4.22, showed that for the highest concentration tested, ⁓97% of

displacement was obtained. For Target 2 μM, used for our analysis, the displacement

114

is ~93%. Decreasing target concentrations up to 500 pM, we reach ~73% of

displacement. Between 400-325 pM displacement decrease at ~40% and for lower

concentrations, no significant displacement were obtained.

Figure 4.22: Displacement percentage for different Target concentrations.

CLSM images of microgels. Scale bars: 100 μm.

To test the assay performance in complex fluids, ds displacement assay was carried

out spiking 2 μM of the target in defined volumes of human serum sample.

Results confirmed an efficient displacement of ~90% also in this case.

Figure 4.23: Fluorescence intensity of (a) T-DNA, (b) adding F-DNA,

(c) displacement in presence of the Target in buffer solution and (d) in human serum.

115

To sum up these CLSM analysis confirm the efficiency of our detection system in

both PBS buffer and Human Serum. Furthermore, preliminarily tests were fulfilled

using a bead-based microfluidic assays. This system showed an edge over normal

fluidic systems, as it employs microbeads as a solid support. In such way, it was

possible to increase the sensitivity of the assay due to higher efficiency of

interactions between samples and reagents. Moreover, the analytes attached onto the

microparticles can be easily transported in a fluidic system using pressure-driven

flow. Microfluidic chip consist of a set of pillars which define a chamber where the

beads are confined for both washing and subsequent analysis steps (Figure 4.24 a).

A thickest set of pillars were arranged before the outlet in order to trap the

microparticles (Figure 4.24 b).

This preliminary test foresee the possibility of using our assay for point-of-care

tasting.

Figure 4.24: Bead-based microfluidic assays with fluorescent microparticles. Scale bars 100 μm.

116

4.4 Conclusions

In this chapter, we focused on the synthesis, characterization and functionalization

of PEGDA-based microparticles. Probe was properly designed for detection of

miRNA-143-3p, responsible of several cardiovascular diseases.

Swelling characterization confirmed results already obtained for bulk hydrogels in

chapter 3. In order to obtain different molecular filter capability, it was possible to

modulate both microfluidic parameters and recipes to generate stable and

monodisperse microparticles, with high control of both size, shape, chemistry and

swelling parameters. In particular, our results pointed out that microparticles

synthesized with PEGDA 15% and T-DNA 1μM best combined both stability and

network structure ability for an efficient hybridization. In particular, highest polymer

concentration show a too narrow network while 10% PEGDA let us to produce too

soft microparticles that are not enough resistant to washing procedure.

Diffusion studies, fulfilled with CLSM, demonstrated the capability of our

microparticles to be used as molecular filter letting target go in and excluding the

majority of big molecules. Finally, our detection system resulted to be very efficient

also in complex fluids as human serum, with a displacement percentage of ⁓90%.

Preliminary experiments, carried out using a bead-based microfluidic assay, showed

the opportunity to improve our system achieving a point-of-care device.

To sum up, our results confirmed the possibility to synthesize engineered and

functionalized hydrogel-based microparticles, with a tunable network, that allow to

combine both molecular filter and detection capability. In this way, we achieve a

new system to simplify the detection of miRNA 143-3p in complex fluids.

117

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Chapter 5

Conclusions and future perspectives

The work described in this thesis intends to show the combined effect of materials

knowledge and biosensing to produce engineered device for detection. In particular,

we achieved a versatile and innovative assay, which successfully combine biomarker

miRNA detection with molecular filter capability of engineered and functionalized

hydrogels with several architectures.

PEG-based hydrogels resulted to be ideal materials for this purpose combining broad

range of molecular weight and chemical functionalities with biocompatibility and

antifouling properties.

Nowadays the detection of biomarkers such as nucleic acids, in particular of

miRNAs, have attracted high attention due to their immense regulatory power so

that the variation of their levels can be used as potential biomarkers for the diagnosis

and prognosis of a variety of diseases.

Firstly, we synthesized and characterized microgels with complex architecture

core-shell. The flexibility in synthesis steps of these fluorescent encoded microgels,

make them suitable for multiplex analysis. Moreover, our results showed the

possibility to attain a high control of size and structural parameters, characterizing

each shell combining both AFM images and equilibrium-swelling theory.

Then, the possibility to functionalize hydrogels directly during synthesis step let us

to find new strategies to eliminate time-consuming labelling steps and their

associated costs.

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In particular, we synthesized bulk hydrogels properly functionalized with

methacrylate oligonucleotide. We demonstrated the possibility to achieve a specific

control of both network structure and efficiency of the capture element, using both

NMR technique and fluorimetric analysis. Moreover, scaling-down this knowledge

into micrometric scale, we produced in a single step, by microfluidic, monodisperse,

spherical and engineered microparticles able for specific detection.

This approach represents the most promising methods for the production and the

functionalization of monodisperse particles at low cost and reagent consumption,

with higher reproducibility and saving time.

In particular, we added directly in flow a probe, properly designed for the detection

of miRNA143-3p (responsible of cardiovascular diseases) and, in such a way, we

were able to detect the specific target also in complex fluids.

In conclusion, we demonstrated the possibility to obtain hydrogels with different

architecture and shape, with high control of both network structure and position of

capture element.

This approach promises to be useful to achieve a novel, efficient and sensitive

hydrogel-based no-wash assay, combining molecular filter capability and

biomarkers detection to simplify the miRNA detection in complex fluids.

The final goal of this project is the production of a microfluidic lab-on-a-chip (LoC)

platform for in vitro, direct measurement of miRNAs present in biological fluids. In

particular, foresee the possibility to accomplish a miniaturized system that combine

both filter system and optical device for fluorescence signal transduction.

The integrated optical device, appropriately optimized, allow merging the

purification, extraction, detection and analysis procedures all inside the device. In

such a way, we can directly analyze a single blood drop overcoming time-consuming

manipulations. As in fact, our major challenge is pointed towards the production of

a device for point-of-care (POC) diagnostics.

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Traditional system do not address the needs of the majority of the world's people

afflicted with infectious diseases, who have, at best, access to poorly resourced health

care facilities with almost no supporting clinical laboratory infrastructure.

The versatility of our system exhibited the possibility to use our engineered

microparticles for the detection of several diseases.

Preliminaries studies showed the potentiality of the system also for the detection of

tuberculosis infection. In such a way, our system may provide some improving in

the actual research on several diseases, improving the quality of life.

Ing. Alessia Mazzarotta - unina.itAlessia Mazzarotta Supervisors: Advisor: Prof. Dr. Paolo Antonio...

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